- Email: [email protected]
Workshop 4: The role of bronchoprovocation
VOLUME NUMBER
2. 3. 4.
5.
6.
78 3. PART 2
Study design
ican Academy of Allergy and Clinical Immunology. San Francisco, Calif: American Academy of Allergy and Clinical Immunology, 1982. ACCP-ATS Joint Committee on Pulmonary Nomenclature: Pulmonary terms and symbols. Chest 1975;67:383. Slavin RG. Relationship of nasal disease and sinusitis to bronchial asthma. Ann Allergy 1982;49:76. General Considerations for the Clinical Evaluation of Drugs in Infants and Children. Washington, DC: US Government Printing Office, 1977; HEW publication No. (FDA)77-3041. General Considerations for the Clinical Evaluation of Drugs. Washington, DC: US Government Printing Office, 1977; HEW publication No. (FDA)77-3040. Welliver RC. Upper respiratory infections in asthma. J ALLERGY
CLIN IMMUNOL1983;72:341.
7. Veterans Administration Cooperative Study Group on Antihypertensive Agents: Effects of treatment on morbidity in hypertension. III. Influence of age, diastolic pressure and prior cardiovascular disease. Circulation 1972;45:99 1. 8. Stevenson DD. Diagnosis, prevention, and treatment of adverse reactions to aspirin and non-steroidal anti-inflammatory drugs. J ALLERGYCLIN IMMUNOL1984;74:617. 9. Szcaekiik A, Gryglewski RJ, Czemiawska-Mysik G. Clinical patterns of hypersensitivity to non-steroidal anti-inflammatory drugs and their pathogenesis. J ALLERGYCLIN IMMUNOL
1977;6(1:276.
Workshop Frederick
10. Jacobs RL, Rahe GW, Foumier DC, Chihon RJ, Culver WG, Beckmann CH. Potentiated anaphylaxis with drug-induced beta adrenergic blockade. J ALLERGYCLINIMMUNOL1983;68:125. 11. Wilson N, Silverman M. Controlled trial of slow release aminophylline in childhood asthma; are short-term trials valid? Br Med J 1982;284:863. 12. Hills M, Armitage P. The two period cross-over clinical trial. Br J Clin Pharmacol 1979;8:7. 13. Shapiro GG, Furukawa CT, Pierson WE, Gardiner R, Bierman CW. Double-blind evaluation of methylprednisolone versus placebo for acute asthma episodes. Pediatrics 1983;71:510. 14. Gould AL. A new approach to the analysis of clinical drug trials with withdrawals. Biometrics 1980;36:721_ 15. Correspondence. Withdrawals from drug trials. Biometrics 1982;38:276. 16. Hudgel DW, Cooperson DM, Kinsman RA. Recognition of added resistive loads in asthma. The importance of behavioral styles. Am Rev Respir Dis 1982;126:121. 17. Kinsman RA, Dahlem NW, Spector S, Staudenmayer H. Observations on subjective symptomatology, coping behavior and medical decisions in asthma. Psychosom Med 1977;39: 102. 18. McFadden ER Jr, Kiser R, DeGroot WJ, et al. Acute bronchial asthma. Relations between clinical and physiologic manifestations. N Engl J Med 1973;288:221. 19. Rubinfield AF, Pain MCF. Perceptions of asthma. Lancet 1976;1:882.
4: The role of bronchoprovocation
E. Hargreave,
Chairperson,
and Jordan
N. Fink, Cochairperson
MEMBERS: Donald W. Cockcroft, James E. Fish, Stephen E. Helen Ramsdale, Robin S. Roberts, Gail G. Shapiro, and Dean Sheppard
I. Introduction II. Rationale for use of provocation tests III. Types of provocation tests and their use A. Inhalation tests with chemical mediators B. Tests with natural nonsensitizing stimuli C. Inhalation tests with allergens IV. Methods of provocation tests A. Patient and environment factors B. Measurement of response C. Chemical mediators D. Exercise E. Hyperventilation F. Allergens V. Safety VI. Examination of effects of drugs VII. summary
INTRODUCTION
Asthma is a diseasein which the airways narrow readily to a variety of stimuli and can therefore be
T. Holgate,
studied by provocation tests with these stimuli. The provocation tests include inhalation tests with chemical mediators of bronchoconstriction, tests with natural nonsensitizing stimuli, and inhalation tests with allergens. Such tests elicit asthmatic responsesthat are early or late in onset. RATIONALE TESTS
FOR USE OF PROVOCATION
Provocation tests provide a valuable adjunct to the evaluation of NBAAD.lm3Such studies provide scientific information concerning the pharmacology of the drugs in humansand, as such, serve as a valuable link between preclinical pharmacologic studies and controlled clinical trials. Because they offer useful information concerning the potential clinical efficacy of drugs, they may be helpful in obtaining information 517
518
Hargreave
J. ALLERGY
et al.
about the optimum dose and duration of effects of NBAAD.4 Extrapolation of thesemodelsto the clinical efficacy of the drugs in the treatmentof asthma,however, must be approachedwith considerablecaution. For example, cromolyn sodium,5o-adrenergic blocking agents6 and calcium channel blockers’ all inhibit exercise-inducedasthma,but only cromolyn hasbeen shown to have any clinical benefit. Corticosteroids have no acute effects on the allergen-induced early asthmatic response but inhibit the allergen-induced late asthmatic responseand remain among the most potent drugs in the treatmentof asthma.’ Conversely, indomethacin, which also inhibits the late but not the early phase of the allergen-induced asthmatic response,‘.9 is of no proved benefit in the treatment of asthma. Hence it is emphasizedthat the use of provocation tests is not a substitute for controlled clinical trials, nor should provocation tests be viewed as a necessarystep in the various stages of drug development. TYPES OF PROVOCATION TESTS AND THEIR USE Inhalation tests with chemical mediators
Inhalation tests with histamine, methacholine, and other cholinergic agonists,“, I1 prostaglandins (PGD* and PGF,,),“, I3 leukotrienes (LTC,, LTD4),14,I5 and adenosine16stimulate early asthmatic responsesthat begin within minutes of their administration. They are considered to act directly on receptors of airway smooth muscle, although other effects may exist. For example, histamine also actson irritant receptors,and the effects of adenosine are inhibited by cromolyn, which suggeststhat mediatorreleaseby this agentmay also be involved. Patientswith asthmatend to be hyperresponsiveto bronchoconstrictormediators.” In general, the greater the responsivenessto one mediator, the greater the responsivenessto another, although exceptionsdo occur. ” The closestcorrelation is betweenthe degreeof responsivenessto histamine and the degree of responsivenessto methacholine. Responsesare usually within a twofold difference, which indicates that the clinical interpretation of results with either agent is the same. The presenceand severity of histamine or methacholine airway hyperresponsivenesscorrelates very closely with the presenceand severity of variable airflow obstruction in asthma.” The degree of responsiveness, and hence the severity of variable airflow obstruction, can be heightened for days, weeks, or months by reactions to inhaled allergens, chemical sensitizers, and, perhaps, respiratory infections and agentssuch as ozone that causeinflammation. Hence
CLIN. IMMUNOL. SEPTEMBER 1986
the degreeof airway responsivenessmeasuredby histamine or methacholine is an important characteristic of asthma. While the airway responsivenesscan be measuredby either agent, there are pros and cons in deciding which one to use. For example, histamine causesairway narrowing of shorterduration than does methacholine” and generally gives the sameresponse when the test is repeatedat short intervals (about 45 minutes).” However, it also produces more systemic side effectswhen higher dosesare required to measure responsivenesswithin the normal range. Inhalation tests with chemical mediators are not representative of natural asthma. Nevertheless, they have a number of specific usesin the investigation of NBAAD: (1) characterizationof the degreeof airway responsivenessin subjectsentering the study; (2) examination of the pharmacologicselectivity of the drug under investigation. For example, the H,-receptor antagonismof ketotifen hasbeeninvestigatedin relation to tbe drug’s effect on airway responseto histamine.3 The effectivenessof a “selective” LTD, antagonist could be examined by its effect on LTD,-stimulated bronchoconstriction; (3) investigation of the nonimmediate effects of drugs in reducing airway responsivenessto histamine or methacholine in asthma”, “; and (4) determination of the effect of drugs on the prevention or reversalof allergen-inducedincreasesin nonallergic airway responsiveness.*’ Tests with
natural
nonsensitizing
stimuli
These tests include exercise and isocapnic hyperventilation of dry air.” Inhalation tests with sulfur dioxide or ultrasonically nebulized water are new tests; a firm understanding of their place in the investigation of drugs requires further experience. Exercise and hyperventilation elicit early asthmatic responses,while on occasionsexercisemay causea late asthmatic response.The responsesare thought to be associatedwith the releaseof bronchoconstrictor mediators into the airways. In general, patients wi.th asthmatend to be hyperresponsiveto these environmental stimuli, and there is a tendency for the degreeof responsivenessto parallel that measuredby histamine and methacholine.” However, it is important to appreciate that each of these stimuli act through one or more different but specific mechanisms,and that differences in the degree of airway responsivenessto them may occur in different individuals. The principal advantagesof exercise challenge are the easeof performance and the natural character of the stimulus, which permits simulation of an environmental situation. However, the major limitation is the impracticality in obtaining a graded stimulus re-
VOLUME NUMBER
78 3 PART 2
The role of bronchoprovocation
sponse curve, which is necessary to define quantitatively the degree of protection afforded by a drug. An advantage of isocapnic hyperventilation is that a stimulus response curve can be easily obtained; however, the range of responsiveness is small and often the degree of inhibition afforded by a drug cannot be quantified precisely unless the initial responsiveness is severely increased.” Inhalation
tests with
allergens
Inhaled allergens will only affect the airways of people who have an allergy. They are common triggers of bronchoconstriction when there is established asthma, and are also primary causes of asthma in people with normal airway responsiveness to histamine or methacholine. They usually stimulate isolated early asthmatic responses or early followed by late asthmatic responses (dual asthmatic responses), which tend to be associated with prolonged increases in airway responsiveness to chemical mediators. 23 Early asthmatic responses begin within minutes, reach a peak within 10 to 30 minutes, and clear within I .5 to 3 hours. They are considered to be chiefly caused by airway smooth muscle constriction as a result of endogenous mediator release. In keeping with this possibility. they are inhibited by B-adrenoceptor agonists and cromolyn sodium.” * The late asthmatic responses usually begin between 3 to 4 hours after allergen exposure, peak between 8 and 12 hours, although occasionally the responses may last longer, and clear within 24 hours. Late responses are associated with prolonged increases in airway responsiveness to histamine or methacholine.24 These are considered to be a result of the cellular phase of inflammation and, besides muscle constriction, they involve other structural changes within the airways, such as edema and sputum production. Late asthmatic responses are inhibited by cromolyn sodium,” * corticosteroids.’ and inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism,8.9 if given before the provocation. Thus the effect of NBAAD can be studied on allergen-induced early and late asthmatic responses, together with the associated prolonged increa.ses in nonallergic airway responsiveness. METHODS
OF PROVOCATION
TESTS
The value of provocation tests is based on the assumption that they represent a controlled reproduction of a clinical event. Their regulation is extremely important but has often been neglected in the past. Regulation requires that the patient, environment, stimulus, and airway measurements be the same for all treatment conditions under study. These requirements
519
have recently been discussed in detail for tests with chemical mediators, exercise, and hyperventilation. ’ ’ There have been recent reviews on methods of allergen inhalation testing.25~x Patient
and environment
factors
These include currently used medications, baseline airway caliber, and recent or current exposure to stimuli that can alter airway responsiveness. Medications that acutely influence the response must be withheld for the duration of their effect. Corticosteroids should be continued at the same dosage throughout the study. Baseline caliber should be similar on each study day and preferably as close to normal as possible. If tests are carried out when airway caliber is reduced, the interpretation of results may be difficult. It is usual to recommend a few weeks’ gap from the last exacerbation of asthma or the last natural exposure to a sensitizer or a respiratory infection that might have heightened airway responsiveness. The tests should be performed at the same time of day because of possible circadian variation in airway responses. Measurement
of response
The FEV, is the simplest and most reproducible measurement used to reflect changes in airway caliber and, when used to measure responsiveness, gives the clearest separation between patients who currently have asthma and those who do not.27 It is therefore suitable for the measurement of responsiveness and the effects of drugs on responsiveness. Of course, other tests can be used, depending on the objectives of the study. The so-called small breath tests such as Raw, SGaw, total lung resistance, and partial expiratory flow volume curves can be more sensitive measures of the response because the full inspiratory maneuver of the FEV, may cause a bronchodilator effect. Hence these tests can be useful for measuring airway responsiveness after a smaller stimulus, for documenting the stimulus response curve in more detail, and for measuring the degree of effect of a drug on responsiveness when this cannot be accurately achieved by study of the FEV,. However, these tests suffer from the disadvantage of greater variance than the FEV, ” In children who are too young (<5 years) to undergo spirometric testing, the PEFR can be used to measure airway narrowing. Children 3 years old and older can often perform this test in a reproducible manner. 28 Chemical
mediators
The dose of aerosol deposited in the lung is determined by the method of aerosol generation and in-
520
Hargreave
J. ALLERGY
et al.
halation. Two methods are now most commonly used. I’ In the method of continuous aerosol generation and tidal breathing, the output of the nebulizer must be measured at regular intervals. Results are more reproducible with tidal breathing for 2 minutes than for 30 seconds. In the dose delivery methods (dosimeter, hand-operated nebulizer), the influence of the volume delivered per puff, the lung volume at delivery (residual volume or functional residual capacity), the speed of inhalation,“’ and how long the breath is held at the end of inspiration need to be investigated. Until their significance is known, each must be specified. The dose delivered per puff must be measured at regular intervals. In both methods the particle size should be measured if possible. While recent studies suggest that variations between about 1 and 5 pm aerodynamic mass median diameter (as produced by most commercial nebulizers) do not appear to influence the response, this point requires further investigation. The possible effects of differences in particle size can be overcome by the use of the same nebulizer in individual patients or studies. The plumbing between the nebulizer and the mouth must be kept constant. In younger children the tidal breathing method is preferable because it does not require coordination between activation of the nebulizer and the beginning of inspiration. All provocation (and drug) solutions should be prepared in isotonic saline solution, because hypotonicity itself is a potent stimulus of bronchoconstriction.” Consideration needs to be given to the stability of various concentrations of the mediator in the diluent that is used to prepare them. Histamine acid phosphate and methacholine chloride in normal saline solution in concentrations between 0.03 and 16 mg/ml are stable when stored at 4” or 21” C for at least 3 months. By contrast, PGD1, LTC, and LTD, must be freshly prepared. 14.” The tests are best carried out in a dose response fashion.” The first inhalation is usually of a normal saline solution control. The number of measurements performed”’ and the one used for analysis (worst, best, mean, or most reproducible) must be specified. If a significant fall in FEV, (15% to 20%) occurs after saline solution, the test should probably be abandoned. If the FEV, is the same as or slightly increased from baseline, the constrictor agonist is then administered cumulatively at intervals of 1 to 5 minutes. The response peaks at different intervals between subjects-for example, the peak responses to histamine or methacholine-may vary between 1 and 4 minutes. Thus the time between inhalations and the time of measurements should be kept constant. The response is usually measured from the postsaline solution mea-
CLIN. IMMUNOL. SEPTEMBER 1986
surements. The inhalations are generally repeated until the FEV, has fallen by 20% or until the results of the small breath tests have changed by 35% to 40%. The results have been best expressed as a point on the steep linear part of the dose response curve, such as PDZo, FEVI, or PDa SGaw.” Expression as the dose delivered to the mouth by the method of aerosol generation and inhalation, rather than the concentration in milligrams per milliliters or cumulative units (which does not include the important measurement of nebulizer output or output per puff ), might make it easier to compare results by the same method between laboratories. However, this needs to be validated. Expression of dose in milligrams or micromoles is suggested. Whether the dose is in cumulative or noncumulative units must be specified. Exercise The major methodologic requirement for both exercise and eucapnic hyperventilation in drug testing is a precise definition of the stimulus in quantitative terms.” Heat and water loss from the airways are the major determinants of the airway response; these are in turn directly related to the level of ventilation and the inspired air temperature and humidity. With exercise the 1eve.lof ventilation is the most important factor if the task is performed under normal ambient air conditions (usually 20” to 26” C; 3 to 12 mg H,O/L). The effect of differences in humidity can be removed by the inhalation of dry air during the exercise. Because the ventilation level is primarily related to the amount of work performed, the intensity and duration of exercise are the major factors to be controlled. Exercise is preferably performed by a motor-driven treadmill, but a cycle ergometer is a suitable alternative. Free running is an unpreditable stimulus. A work load equivalent to 70% to 80% of the predicted maximum oxygen consumption, sustained for an interval of 6 to 8 minutes, is usually optimal to achieve and maintain ventilation at a level sufficient to provoke airway narrowing in most patients with asthma. For proper control and documentation, the following information should be recorded during exercise challenges: (1) inspired air temperature and water content; (2) minute ventilation; (3) duration of exercise; (4) exercise work load; and (5) heart rate. The intensity of the work load can be confirmed by the heart rate because of the correlation between maximum oxygen consumption and heart rate. Heart rate should reach approximately 90% of the age-predicted maximum (approximately 2 10 beats per minute minus age [in years] in adults or as indicated by the reference nomogram in children). Moreover, monitoring the heart rate provides a measure of safety for the patient
VOLUME NUMBER
78 3, PART 2
and permits adjustment of work load for differences in fitness levels between subjects. Measurements of the response should be made at rest, after the drug just before the test, and after the test at specified intervals for at least 15 minutes. The response to exercise may be expressed as the percent fall from the value immediately before exercise. This so-called “percent fall index” is of value only if prechallenge measurements are at comparable levels on different test days. Hyperventilation The major variables to be controlled include the following: (1) level of ventilation; (2) duration of hyperventilation; (3) inspired air temperature; and (4) water content of inspired air.” The challenge is performed by having patients voluntarily hyperventilate with incompletely conditioned air, usually dry gas, which may also be cooled to - 10” to -40” C. Expired air is passed through a carbon dioxide meter to measure partial pressure of carbon dioxide. Eucapnic conditions are maintained by adding carbon dioxide to the inspired gas at a rate sufficient to maintain end tidal partial pressures between 38 and 42 mm Hg. The procedure may be simplified by having patients inspire dry gas at room temperature, thus obviating the need for a heat exchanger.3’ At room temperature hyperventilation of dry gas at ventilation levels comparable to those achieved during strenuous exercise (60 to 70 L/min) can trigger airway narrowing in most patients with asthma. Cooling of the inspired air can potentiate the response. The optimum duration of hyperventilation is between 3 to 4 minutes. By varying the level of the stimulus intensity, one can use eucapnic hyperventilation to obtain graded stimulus response curves. This provides a means of evaluating the effects of a drug on the response to increasing levels of stimulus intensity, thus providing more complete information on the degree of protection. The use of eucapnic hyperventilation challenges to obtain stimulus response curves was based on earlier observations that hyperpnea failed to induce a refractory period similar to exercise. More recent studies, however, have demonstrated refractoriness after hyperpnea. It seems clear, however, that graded stimulus response curves can be obtained with eucapnic hyperventilation despite the occurrence of refractoriness, and that the reproducibility of such curves is adequate for drug testing. The most common practice is to vary the stimulus intensity by changing the minute ventilation level at fixed inspired air conditions. Varying levels of ventilation in onefold or twofold increments, starting at 7.5 L/mm and increasing to maximum voluntary ventilation, can be performed in
The role of bronchoprovocation
521
sequence, allowing for a brief interval between challenges for measurement of the response. The response is plotted against the stimulus intensity. The stimulus intensity is most simply expressed as the minute ventilation provided that inspired air conditions are kept constant at different levels of hyperpnea. Alternatively, the stimulus can be expressed in terms of units of respiratory heat exchange. However, the use of these units as an expression of stimulus intensity is somewhat controversial because of either unproved assumptions concerning expired air conditions or technical difficulties in actual measurements of expired air temperature. Calculations of respiratory heat exchange in many early studies were often based on assumed values of expired air temperature and humidity rather than actual measurements. The assumption that expired air is completely saturated under all temperature conditions has been questioned, and further experiments are needed to confirm this assumption. Furthermore, because of the limited response times and calibration problems of most commercially available temperature recording devices, the precision of expired air temperature measurements has been questioned. At present, minute ventilation is the preferred expression of the stimulus intensity. Units of respiratory heat exchange are also considered acceptable, provided that the investigators acknowledge whether expired air temperature and humidity are in fact measured or whether assumed values are used. The airways response to the graded stimulus is measured before drug administration, after administration just before the test, and after the test at l-minute and 2 to 3-minute intervals until the peak of the response is obtained. Allergens The principles for regulation of allergen inhalation tests are similar to those for inhalation tests with chemical mediators.25.26Differences result from problems with standardization of test extracts, the dependence of the early asthmatic response on the level of IgE antibodies, airway responsiveness to endogenous release of chemical mediators and the dose of allergen, the different time course of the early asthmatic response that peaks later, and the frequent occurrence of late asthmatic responses with an associated prolonged increase in airway responsiveness. Crude allergen extracts are not well characterized with respect to the number and concentration of antigens they contain. Purified allergens, such as antigen E derived from crude ragweed extract, are available for some exogenous causes of asthma. In any case, there must be enough of the same extract lot for each
522
Hargreave
et al.
subject in the study. Furthermore, crude or purified allergen extracts degrade with time. The degradation can be reduced by storage between 4” and - 20” C and by the addition of a stabilizer such as human serum albumin. When the effect of a drug on the allergeninduced response is examined, allergen extracts should be stored at - 20” C in freeze-dried form or as the concentrate in single-use aliquots. The extracts should be reconstituted or thawed and diluted shortly before use on the same day. They should not activate complement nonspecifically. The allergen-induced early asthmatic response is chiefly determined by the level of specific IgE antibodies, the dose of antigen, and the level of airway responsiveness to histamine or methacholine.23 The first two factors determine the presence and degree of mediator release and the latter indicates the effect of the mediators on airway structures. At the start of the study, threshold allergy skin tests are required to determine a safe dose to begin allergen inhalations.23 The allergen-induced early asthmatic response is usually advanced at 10 minutes and peaks between 10 and 30 minutes. As a result, measurements of the response are conveniently made at about 10 minutes. Allergen inhalations are stepped up until there is a response equivalent in severity to a fall in FEV, of about 15% to 20%. Convenient times to follow the early response are 5, 10, 15, 20, and 30 minutes and 1, 1.5, 2, and 3 hours. When repeat tests are made, the starting dose of allergen should be at least two twofold doses below the final dose in the first test. Late asthmatic responses commonly occur after allergen inhalation and must be recorded by the repetition of measurements each hour for at least 8 hours after the allergen-induced early response. Their severity cannot yet be predicted, although three important determinants have been indicated: ( 1) the dose of allergen, (2) the severity of the early asthmatic response, and (3) the baseline histamine or methacholine airway responsiveness. Their association with prolonged increases in histamine or methacholine airway responsiveness may make it impossible to repeat allergen inhalation tests until the hyperresponsive state has returned to baseline (for 1 or more weeks).
SAFETY The primary consideration in the use of provocation test protocols must be the protection of the health and safety of the subjects. Because of the potential risks associated with the challenge procedure itself, studies should be carried out only in patients who have been appropriately screened and found suitable on the basis of clinical status and pulmonary function stability. Moreover, because of the potential for severe and un-
J ALLERGY
CLIN. IMMUNOL. SEPTEMBER 1986
expected reactions to allergens, it is recommended that allergen inhalation testing be performed only by investigators experienced in these procedures in a setting in which untoward reactions can be anticipated and properly managed. When these tests are used in the evaluation of drug efficacy, proper informed consent for challenge protocols must be reviewed and approved by institutional review boards.
EXAMINATION OF EFFECTS OF DRUGS The same scientific principles that apply to the study design and analysis of all drug trials, specifically regarding definition of the study question, treatment controls, blinding, randomization, and so forth, must also be applied to provocation test studies. A number of additional points particularly relevant to provocation tests are discussed here. The reproducibility of responses in each laboratory should be known and should match the expected reproducibility. Reproducibility can vary between individuals and studies and therefore, if there is doubt, it should be assessed in the study design. The response to the test can be altered by the previous test, and therefore consideration should be given to the interval between tests to try to avoid this possibility. For example, the response to exercise can be followed by a refractory period for 2 to 3 hours during which the response to another exercise test will be reduced. It is therefore recommended that exercise tests be repeated at intervals of 3 to 4 hours or more to avoid this possibility. The best method to examine the magnitude of effect of a drug on early asthmatic responses is to measure the change in position of the stimulus response curve. This is possible in all provocation tests with the exception of exercise, for which stimulus response curve determinations take too long to be practical. When allergen-induced early responses are examined in this way, there is the possibility of causing a subsequent severe late asthmatic response if the drug inhibits the former response more than the latter. Therefore, the patient must be carefully followed for this possibility or treated to prevent it. It is also recommended that a time course study be performed to determine the optimum time zt which a drug might be expected to exert its effect after the allergen challenge. For example, an H,-receptor antihistamine should be evaluated within 5 minutes after the challenge, because histamine plays a major role in immediate bronchoconstriction. On the other hand, the optimal time to examine an anti-LTD, effect is between 10 and 30 minutes after challenge, because membrane-generated mediators account for most of the bronchoconstrictor effect at this time.
VOLUME NUMBER
The role of bronchoprovocation
78 3. PART 2
Examination of the effect of drugs on allergen-induced early and late asthmatic responses and increases in airway responsiveness can be important to examine, but the best approach to the study design needs investigation. However, at present there are too many uncertainties about the variables that influence results to be able to draw up guidelines on a factual basis. Nevertheless, it is felt that these gaps in our understanding should not be a deterrent to studies of drug effects on allergen-induced responses. Rather, carefully designed protocols to investigate drug effects in this model of acute airway inflammation are encouraged as a means of advancing our understanding of drug effects as well as the disease we seek to treat. Baseline airway caliber can influence results. This is less likely in subjects with normal baseline measurements and in short-term studies with NBAAD. In long-term studies, however, these drugs can produce bronchodilation as a secondary event. At present, it is not known how to analyze the effect of a change in airway caliber, but absolute predrug and postdrug values, predicted values, and percentage changes should all be reported (see workshop 5). One method recently used to investigate the effect of change in airway caliber has been to adjust for the change by analysis of covariance.32 The analysis of results of PD, or SGaw, should be carried out on log-transformed data. Almost all statistical procedures require that the inherent variability of an outcome measure be constant over subjects or groups of subjects. This is not true for PD,, or SGaw,. for which individual values from different patients may differ by two logs or more. A logarithmic transformation (either natural or to base 10) of PDZO and SGaw, eliminates the tendency for standard deviation to be related to the mean. All statistical comparisons should thus be conducted after first performing log transformation of individual values. Summary statistics can be quoted either on the transformed scale or after conversion to a linear scale. Although the logarithmic transformation is used here primarily to stabilize variance, it will also tend to produce a more normal distribution and perhaps linearize relationships with other factors such as drug dose. SUMMARY Provocation tests can be. used to investigate the effects of NBAAD. These studies provide scientific information on the pharmacology of drugs in humans, their potential clinical efficacy, and the optimum dose and duration of their effect. They serve as a useful link between preclinical studies and clinical trials; however, they do not necessarily identify the clinical value of drugs and they are not a substitute for clinical
523
trials. Provocation tests include inhalation tests with chemical mediators of airway narrowing, tests with natural nonsensitizing stimuli such as exercise and hyperventilation, and inhalation tests with allergens. Each of these stimuli acts through different specific mechanisms. All of them can trigger early asthmatic responses; exercise can also stimulate late asthmatic responses; and allergens can induce late asthmatic responses and prolonged increases in airway responsiveness to chemical mediators. The provocation tests must be carefully regulated to produce reproducible responses against which the effect of the drugs can be examined. This stipulation requires that the patient, environment, stimulus, and airways measurements are the same for each treatment condition. The study design must incorporate the scientific principles applicable to all drug studies. While guidelines can be given to studies that involve tests with chemical mediators, exercise, and hyperventilation, they cannot yet be specified for allergen inhalation tests, for which the factors that influence reproducibility are less well understood. REFERENCES 1. Pepys J, Chan M, Hargreave FE, McCarthy DS. Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 1968;2: 134-7. 2. Booij-Noord H, Orie NGM, de Vries K. Immediate and late bronchial obstructive reactions to inhalation of house dust and protective effects of disodium cromoglycate and prednisolone. J ALLERGY CLIN IMMUE~OL1971;48:344-54. 3. Phillips MJ, Oilier S, Gould CM, Davies RJ. Effect of antihistamines and antiallergic drugs on responses to allergen and histamine provocation tests in asthma. Thorax 1984;39:34551. 4. Juniper EF, Latimer KM, Morris MM, Roberts RS, Hargreave FE. Airway responses to hyperventilation of cold dry air: duration of protection by cromolyn sodium. J ALLERGY CLIN IMMUNOL 1986;78:387-!)l. 5. Godfrey S, Konig P. Inhibition of exercise-induced asthma by different pharmacologic pathways. Thorax 1976;3 1: 137-43. 6. Barnes PJ, Wilson NM, Vickers H. Prazosin, an alpha,adrenoceptor antagonist, partially inhibits exercise-induced asthma. J ALLERGY CLIN IMMUNOL 198 1;68:4 11-5. 7. Conis PA, Nariman S, Gibson GJ. Nifedipine in the prevention of asthma induced by exercise and histamine. Am Rev Respir Dis 1983;128:991-2. 8. Fairfax AJ, Hanson JM, Morley I. The late reaction following bronchial provocation with house dust mite allergen. Dependence on arachidonic acid metabolism. Clin Exp Immunol 1983;52:393-8. 9. Joubert JR, Shephard E, Mouton W, Van Zyl L, Viljoen I. Non-steroid anti-inflammatory drugs in asthma: dangerous or useful therapy? Allergy 1985;40:202-7. 10. Hargreave FE, Sterk PJ, Ramsdale EH, Dolovich J, Zamel N. Inhalation challenge tests and airway responsiveness in man. Chest 1985;87:S202-6. 11. Hargreave FE, Woolcock AJ. Airway responsiveness: measurement and interpretation. Mississauga: Astra Pharmaceuticals Canada Ltd. 1985.
524
Hargreave
J. ALLERGY
et al.
12. Hardy CC, Robinson S, Tatterfield AE, Holgate ST. The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men. N Engl J Med 1984;311:209-13. 13. Thomson NC, Roberts R, Bandouvakis J, Newball H, Hargreave FE. Comparison of bronchial responses to prostaglandin F, and methacholine. J ALLERGY CLIN IMMUNOL 1981;68: 392-8. 14. Adelroth E, Morris MM, Hargreave FE, O’Byme PM. Airway responsiveness and relative potency of leukohienes C,, D, and methacholine in asthmatics and normals. N Engl J Med (in press). 15. Griffin M, Weiss SW, Leitch AG, et al. Effects of leukotriene D on the airways in athma. N Engl J Med 1983;308:436-9. 16. Cushley MJ, Church MK, Pao GJ-K, Holgate ST. The role of mast cell mediator release in adenosine-induced bronchoconstriction. J ALLERGYCLIN IMMUNOL 1985;75:272-8. 17. Cartier A, Malo JL, Begin P, Sestier M, Martin RR. Time course of the bronchoconstriction induced by inhaled histamine and methacholine. J Appl Physiol 1983;54:821-6. 18. Ruffin RE, Alpers JH, Crockett AJ, Hamilton R. Repeated histamine inhalation tests in asthmatic patients. J ALLERGY CLIN IMMUNOL 1981;64:285-9. 19. Ryan G, Latimer KM, Juniper EF, Roberts RS, Hargreave FE. Effect of beclomethasone dipropionate on bronchial responsiveness to histamine in controlled nonsteroid-dependent asthma. J ALLERGY CLIN IMMUNOL 1985;75:25-30. 20. Lowhagen 0, Rak S. Modification of bronchial hyperreactivity after treatment with sodium cromoglycate during pollen season. J ALLERGY CLIN IMMUNOL 1985;75:460-7. 21. Sotomayer H, Badier M, Vervloet D, Orehek J. Seasonal increase in carbachol airway responsiveness in patients allergic to grass pollen: reversal by corticosteroids. Am Rev Respir Dis 1984;130:56-8. 22. O’Byme PM, Morris M, Roberts R, Hargreave FE. Inhibition
23.
24.
25. 26.
27.
28. 29.
30. 3 1. 32.
CLIN. IMMUNOL. SEPTEMBER 1986
of the bronchial response to respiratory heat exchange by increasing doses of terbutaline sulphate. Thorax 1982;37:913-7. Hargreave FE, Frith PA, Dolovich M, et al. Allergen-induced airway responses and relationships with nonspecific reactivity. In: Hargreave FE, ed. Airway reactivity: mechanisms and clinical relevance. Mississauga: Astra Pharmaceuticals Canada Ltd, 1980:145-50. Hargreave FE, Dolovich J. Bronchial responsiveness and late asthmatic response. In: Kay AB, Lichtenstein LM, Austen KF, eds. Asthma: physiology, immunopharmacology and treatment. London: Academic Press, 1984:263-77. Hargreave FE, Dolovich J. Boulet L-P. Inhalation provocation tests. Semin Respir Med 1983;4:224-35. Davies RJ, Corrado OJ. Diagnostic tests-challenge tests: oral, nasal and bronchial. In: Lessof MH, ed. Allergy, immunological and clinical aspects. Chichester: John Wiley & Sons, 198483-105. Fish JE, Kelly JF. Measurements of responsiveness in bronchoprovocation testing. J ALLERGY CLIN IMMUNOL 1979;64: 592-6. Godfrey S. Childhood asthma. In: Clark TJH, Godfrey S, eds. Asthma. 2nd ed. London: Chapman and Hall, 1983:415-56. Laube BL, Adams GK, Norman PS, Rosenthal RR. The effect of inspiratory flow rate regulation on nebulizer output and on human airway response to methacholine aerosol. J ALLERGY CLIN IMM~JNOL1985;76:708-13. Scott GC, Kung M. How many spirograms for a histamine challenge? Am Rev Respir Dis 1985;132:268-71. Rosenthal RR. Simplified eucapnic voluntary hyperventilation challenge. J ALLERGYCLM IMMUNOL 1984;73:676-9. O’Bryne PM, Thomson NC, Morris M, Roberts RS, Daniel EE, Hargreave FE. The protective effect of inhaled chlorpheniramine and atropine on bronchoconstriction stimulated by airway cooling. Am Rev Respir Dis 1983;128:611-7.
2. 3. 4.
5.
6.
78 3. PART 2
Study design
ican Academy of Allergy and Clinical Immunology. San Francisco, Calif: American Academy of Allergy and Clinical Immunology, 1982. ACCP-ATS Joint Committee on Pulmonary Nomenclature: Pulmonary terms and symbols. Chest 1975;67:383. Slavin RG. Relationship of nasal disease and sinusitis to bronchial asthma. Ann Allergy 1982;49:76. General Considerations for the Clinical Evaluation of Drugs in Infants and Children. Washington, DC: US Government Printing Office, 1977; HEW publication No. (FDA)77-3041. General Considerations for the Clinical Evaluation of Drugs. Washington, DC: US Government Printing Office, 1977; HEW publication No. (FDA)77-3040. Welliver RC. Upper respiratory infections in asthma. J ALLERGY
CLIN IMMUNOL1983;72:341.
7. Veterans Administration Cooperative Study Group on Antihypertensive Agents: Effects of treatment on morbidity in hypertension. III. Influence of age, diastolic pressure and prior cardiovascular disease. Circulation 1972;45:99 1. 8. Stevenson DD. Diagnosis, prevention, and treatment of adverse reactions to aspirin and non-steroidal anti-inflammatory drugs. J ALLERGYCLIN IMMUNOL1984;74:617. 9. Szcaekiik A, Gryglewski RJ, Czemiawska-Mysik G. Clinical patterns of hypersensitivity to non-steroidal anti-inflammatory drugs and their pathogenesis. J ALLERGYCLIN IMMUNOL
1977;6(1:276.
Workshop Frederick
10. Jacobs RL, Rahe GW, Foumier DC, Chihon RJ, Culver WG, Beckmann CH. Potentiated anaphylaxis with drug-induced beta adrenergic blockade. J ALLERGYCLINIMMUNOL1983;68:125. 11. Wilson N, Silverman M. Controlled trial of slow release aminophylline in childhood asthma; are short-term trials valid? Br Med J 1982;284:863. 12. Hills M, Armitage P. The two period cross-over clinical trial. Br J Clin Pharmacol 1979;8:7. 13. Shapiro GG, Furukawa CT, Pierson WE, Gardiner R, Bierman CW. Double-blind evaluation of methylprednisolone versus placebo for acute asthma episodes. Pediatrics 1983;71:510. 14. Gould AL. A new approach to the analysis of clinical drug trials with withdrawals. Biometrics 1980;36:721_ 15. Correspondence. Withdrawals from drug trials. Biometrics 1982;38:276. 16. Hudgel DW, Cooperson DM, Kinsman RA. Recognition of added resistive loads in asthma. The importance of behavioral styles. Am Rev Respir Dis 1982;126:121. 17. Kinsman RA, Dahlem NW, Spector S, Staudenmayer H. Observations on subjective symptomatology, coping behavior and medical decisions in asthma. Psychosom Med 1977;39: 102. 18. McFadden ER Jr, Kiser R, DeGroot WJ, et al. Acute bronchial asthma. Relations between clinical and physiologic manifestations. N Engl J Med 1973;288:221. 19. Rubinfield AF, Pain MCF. Perceptions of asthma. Lancet 1976;1:882.
4: The role of bronchoprovocation
E. Hargreave,
Chairperson,
and Jordan
N. Fink, Cochairperson
MEMBERS: Donald W. Cockcroft, James E. Fish, Stephen E. Helen Ramsdale, Robin S. Roberts, Gail G. Shapiro, and Dean Sheppard
I. Introduction II. Rationale for use of provocation tests III. Types of provocation tests and their use A. Inhalation tests with chemical mediators B. Tests with natural nonsensitizing stimuli C. Inhalation tests with allergens IV. Methods of provocation tests A. Patient and environment factors B. Measurement of response C. Chemical mediators D. Exercise E. Hyperventilation F. Allergens V. Safety VI. Examination of effects of drugs VII. summary
INTRODUCTION
Asthma is a diseasein which the airways narrow readily to a variety of stimuli and can therefore be
T. Holgate,
studied by provocation tests with these stimuli. The provocation tests include inhalation tests with chemical mediators of bronchoconstriction, tests with natural nonsensitizing stimuli, and inhalation tests with allergens. Such tests elicit asthmatic responsesthat are early or late in onset. RATIONALE TESTS
FOR USE OF PROVOCATION
Provocation tests provide a valuable adjunct to the evaluation of NBAAD.lm3Such studies provide scientific information concerning the pharmacology of the drugs in humansand, as such, serve as a valuable link between preclinical pharmacologic studies and controlled clinical trials. Because they offer useful information concerning the potential clinical efficacy of drugs, they may be helpful in obtaining information 517
518
Hargreave
J. ALLERGY
et al.
about the optimum dose and duration of effects of NBAAD.4 Extrapolation of thesemodelsto the clinical efficacy of the drugs in the treatmentof asthma,however, must be approachedwith considerablecaution. For example, cromolyn sodium,5o-adrenergic blocking agents6 and calcium channel blockers’ all inhibit exercise-inducedasthma,but only cromolyn hasbeen shown to have any clinical benefit. Corticosteroids have no acute effects on the allergen-induced early asthmatic response but inhibit the allergen-induced late asthmatic responseand remain among the most potent drugs in the treatmentof asthma.’ Conversely, indomethacin, which also inhibits the late but not the early phase of the allergen-induced asthmatic response,‘.9 is of no proved benefit in the treatment of asthma. Hence it is emphasizedthat the use of provocation tests is not a substitute for controlled clinical trials, nor should provocation tests be viewed as a necessarystep in the various stages of drug development. TYPES OF PROVOCATION TESTS AND THEIR USE Inhalation tests with chemical mediators
Inhalation tests with histamine, methacholine, and other cholinergic agonists,“, I1 prostaglandins (PGD* and PGF,,),“, I3 leukotrienes (LTC,, LTD4),14,I5 and adenosine16stimulate early asthmatic responsesthat begin within minutes of their administration. They are considered to act directly on receptors of airway smooth muscle, although other effects may exist. For example, histamine also actson irritant receptors,and the effects of adenosine are inhibited by cromolyn, which suggeststhat mediatorreleaseby this agentmay also be involved. Patientswith asthmatend to be hyperresponsiveto bronchoconstrictormediators.” In general, the greater the responsivenessto one mediator, the greater the responsivenessto another, although exceptionsdo occur. ” The closestcorrelation is betweenthe degreeof responsivenessto histamine and the degree of responsivenessto methacholine. Responsesare usually within a twofold difference, which indicates that the clinical interpretation of results with either agent is the same. The presenceand severity of histamine or methacholine airway hyperresponsivenesscorrelates very closely with the presenceand severity of variable airflow obstruction in asthma.” The degree of responsiveness, and hence the severity of variable airflow obstruction, can be heightened for days, weeks, or months by reactions to inhaled allergens, chemical sensitizers, and, perhaps, respiratory infections and agentssuch as ozone that causeinflammation. Hence
CLIN. IMMUNOL. SEPTEMBER 1986
the degreeof airway responsivenessmeasuredby histamine or methacholine is an important characteristic of asthma. While the airway responsivenesscan be measuredby either agent, there are pros and cons in deciding which one to use. For example, histamine causesairway narrowing of shorterduration than does methacholine” and generally gives the sameresponse when the test is repeatedat short intervals (about 45 minutes).” However, it also produces more systemic side effectswhen higher dosesare required to measure responsivenesswithin the normal range. Inhalation tests with chemical mediators are not representative of natural asthma. Nevertheless, they have a number of specific usesin the investigation of NBAAD: (1) characterizationof the degreeof airway responsivenessin subjectsentering the study; (2) examination of the pharmacologicselectivity of the drug under investigation. For example, the H,-receptor antagonismof ketotifen hasbeeninvestigatedin relation to tbe drug’s effect on airway responseto histamine.3 The effectivenessof a “selective” LTD, antagonist could be examined by its effect on LTD,-stimulated bronchoconstriction; (3) investigation of the nonimmediate effects of drugs in reducing airway responsivenessto histamine or methacholine in asthma”, “; and (4) determination of the effect of drugs on the prevention or reversalof allergen-inducedincreasesin nonallergic airway responsiveness.*’ Tests with
natural
nonsensitizing
stimuli
These tests include exercise and isocapnic hyperventilation of dry air.” Inhalation tests with sulfur dioxide or ultrasonically nebulized water are new tests; a firm understanding of their place in the investigation of drugs requires further experience. Exercise and hyperventilation elicit early asthmatic responses,while on occasionsexercisemay causea late asthmatic response.The responsesare thought to be associatedwith the releaseof bronchoconstrictor mediators into the airways. In general, patients wi.th asthmatend to be hyperresponsiveto these environmental stimuli, and there is a tendency for the degreeof responsivenessto parallel that measuredby histamine and methacholine.” However, it is important to appreciate that each of these stimuli act through one or more different but specific mechanisms,and that differences in the degree of airway responsivenessto them may occur in different individuals. The principal advantagesof exercise challenge are the easeof performance and the natural character of the stimulus, which permits simulation of an environmental situation. However, the major limitation is the impracticality in obtaining a graded stimulus re-
VOLUME NUMBER
78 3 PART 2
The role of bronchoprovocation
sponse curve, which is necessary to define quantitatively the degree of protection afforded by a drug. An advantage of isocapnic hyperventilation is that a stimulus response curve can be easily obtained; however, the range of responsiveness is small and often the degree of inhibition afforded by a drug cannot be quantified precisely unless the initial responsiveness is severely increased.” Inhalation
tests with
allergens
Inhaled allergens will only affect the airways of people who have an allergy. They are common triggers of bronchoconstriction when there is established asthma, and are also primary causes of asthma in people with normal airway responsiveness to histamine or methacholine. They usually stimulate isolated early asthmatic responses or early followed by late asthmatic responses (dual asthmatic responses), which tend to be associated with prolonged increases in airway responsiveness to chemical mediators. 23 Early asthmatic responses begin within minutes, reach a peak within 10 to 30 minutes, and clear within I .5 to 3 hours. They are considered to be chiefly caused by airway smooth muscle constriction as a result of endogenous mediator release. In keeping with this possibility. they are inhibited by B-adrenoceptor agonists and cromolyn sodium.” * The late asthmatic responses usually begin between 3 to 4 hours after allergen exposure, peak between 8 and 12 hours, although occasionally the responses may last longer, and clear within 24 hours. Late responses are associated with prolonged increases in airway responsiveness to histamine or methacholine.24 These are considered to be a result of the cellular phase of inflammation and, besides muscle constriction, they involve other structural changes within the airways, such as edema and sputum production. Late asthmatic responses are inhibited by cromolyn sodium,” * corticosteroids.’ and inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism,8.9 if given before the provocation. Thus the effect of NBAAD can be studied on allergen-induced early and late asthmatic responses, together with the associated prolonged increa.ses in nonallergic airway responsiveness. METHODS
OF PROVOCATION
TESTS
The value of provocation tests is based on the assumption that they represent a controlled reproduction of a clinical event. Their regulation is extremely important but has often been neglected in the past. Regulation requires that the patient, environment, stimulus, and airway measurements be the same for all treatment conditions under study. These requirements
519
have recently been discussed in detail for tests with chemical mediators, exercise, and hyperventilation. ’ ’ There have been recent reviews on methods of allergen inhalation testing.25~x Patient
and environment
factors
These include currently used medications, baseline airway caliber, and recent or current exposure to stimuli that can alter airway responsiveness. Medications that acutely influence the response must be withheld for the duration of their effect. Corticosteroids should be continued at the same dosage throughout the study. Baseline caliber should be similar on each study day and preferably as close to normal as possible. If tests are carried out when airway caliber is reduced, the interpretation of results may be difficult. It is usual to recommend a few weeks’ gap from the last exacerbation of asthma or the last natural exposure to a sensitizer or a respiratory infection that might have heightened airway responsiveness. The tests should be performed at the same time of day because of possible circadian variation in airway responses. Measurement
of response
The FEV, is the simplest and most reproducible measurement used to reflect changes in airway caliber and, when used to measure responsiveness, gives the clearest separation between patients who currently have asthma and those who do not.27 It is therefore suitable for the measurement of responsiveness and the effects of drugs on responsiveness. Of course, other tests can be used, depending on the objectives of the study. The so-called small breath tests such as Raw, SGaw, total lung resistance, and partial expiratory flow volume curves can be more sensitive measures of the response because the full inspiratory maneuver of the FEV, may cause a bronchodilator effect. Hence these tests can be useful for measuring airway responsiveness after a smaller stimulus, for documenting the stimulus response curve in more detail, and for measuring the degree of effect of a drug on responsiveness when this cannot be accurately achieved by study of the FEV,. However, these tests suffer from the disadvantage of greater variance than the FEV, ” In children who are too young (<5 years) to undergo spirometric testing, the PEFR can be used to measure airway narrowing. Children 3 years old and older can often perform this test in a reproducible manner. 28 Chemical
mediators
The dose of aerosol deposited in the lung is determined by the method of aerosol generation and in-
520
Hargreave
J. ALLERGY
et al.
halation. Two methods are now most commonly used. I’ In the method of continuous aerosol generation and tidal breathing, the output of the nebulizer must be measured at regular intervals. Results are more reproducible with tidal breathing for 2 minutes than for 30 seconds. In the dose delivery methods (dosimeter, hand-operated nebulizer), the influence of the volume delivered per puff, the lung volume at delivery (residual volume or functional residual capacity), the speed of inhalation,“’ and how long the breath is held at the end of inspiration need to be investigated. Until their significance is known, each must be specified. The dose delivered per puff must be measured at regular intervals. In both methods the particle size should be measured if possible. While recent studies suggest that variations between about 1 and 5 pm aerodynamic mass median diameter (as produced by most commercial nebulizers) do not appear to influence the response, this point requires further investigation. The possible effects of differences in particle size can be overcome by the use of the same nebulizer in individual patients or studies. The plumbing between the nebulizer and the mouth must be kept constant. In younger children the tidal breathing method is preferable because it does not require coordination between activation of the nebulizer and the beginning of inspiration. All provocation (and drug) solutions should be prepared in isotonic saline solution, because hypotonicity itself is a potent stimulus of bronchoconstriction.” Consideration needs to be given to the stability of various concentrations of the mediator in the diluent that is used to prepare them. Histamine acid phosphate and methacholine chloride in normal saline solution in concentrations between 0.03 and 16 mg/ml are stable when stored at 4” or 21” C for at least 3 months. By contrast, PGD1, LTC, and LTD, must be freshly prepared. 14.” The tests are best carried out in a dose response fashion.” The first inhalation is usually of a normal saline solution control. The number of measurements performed”’ and the one used for analysis (worst, best, mean, or most reproducible) must be specified. If a significant fall in FEV, (15% to 20%) occurs after saline solution, the test should probably be abandoned. If the FEV, is the same as or slightly increased from baseline, the constrictor agonist is then administered cumulatively at intervals of 1 to 5 minutes. The response peaks at different intervals between subjects-for example, the peak responses to histamine or methacholine-may vary between 1 and 4 minutes. Thus the time between inhalations and the time of measurements should be kept constant. The response is usually measured from the postsaline solution mea-
CLIN. IMMUNOL. SEPTEMBER 1986
surements. The inhalations are generally repeated until the FEV, has fallen by 20% or until the results of the small breath tests have changed by 35% to 40%. The results have been best expressed as a point on the steep linear part of the dose response curve, such as PDZo, FEVI, or PDa SGaw.” Expression as the dose delivered to the mouth by the method of aerosol generation and inhalation, rather than the concentration in milligrams per milliliters or cumulative units (which does not include the important measurement of nebulizer output or output per puff ), might make it easier to compare results by the same method between laboratories. However, this needs to be validated. Expression of dose in milligrams or micromoles is suggested. Whether the dose is in cumulative or noncumulative units must be specified. Exercise The major methodologic requirement for both exercise and eucapnic hyperventilation in drug testing is a precise definition of the stimulus in quantitative terms.” Heat and water loss from the airways are the major determinants of the airway response; these are in turn directly related to the level of ventilation and the inspired air temperature and humidity. With exercise the 1eve.lof ventilation is the most important factor if the task is performed under normal ambient air conditions (usually 20” to 26” C; 3 to 12 mg H,O/L). The effect of differences in humidity can be removed by the inhalation of dry air during the exercise. Because the ventilation level is primarily related to the amount of work performed, the intensity and duration of exercise are the major factors to be controlled. Exercise is preferably performed by a motor-driven treadmill, but a cycle ergometer is a suitable alternative. Free running is an unpreditable stimulus. A work load equivalent to 70% to 80% of the predicted maximum oxygen consumption, sustained for an interval of 6 to 8 minutes, is usually optimal to achieve and maintain ventilation at a level sufficient to provoke airway narrowing in most patients with asthma. For proper control and documentation, the following information should be recorded during exercise challenges: (1) inspired air temperature and water content; (2) minute ventilation; (3) duration of exercise; (4) exercise work load; and (5) heart rate. The intensity of the work load can be confirmed by the heart rate because of the correlation between maximum oxygen consumption and heart rate. Heart rate should reach approximately 90% of the age-predicted maximum (approximately 2 10 beats per minute minus age [in years] in adults or as indicated by the reference nomogram in children). Moreover, monitoring the heart rate provides a measure of safety for the patient
VOLUME NUMBER
78 3, PART 2
and permits adjustment of work load for differences in fitness levels between subjects. Measurements of the response should be made at rest, after the drug just before the test, and after the test at specified intervals for at least 15 minutes. The response to exercise may be expressed as the percent fall from the value immediately before exercise. This so-called “percent fall index” is of value only if prechallenge measurements are at comparable levels on different test days. Hyperventilation The major variables to be controlled include the following: (1) level of ventilation; (2) duration of hyperventilation; (3) inspired air temperature; and (4) water content of inspired air.” The challenge is performed by having patients voluntarily hyperventilate with incompletely conditioned air, usually dry gas, which may also be cooled to - 10” to -40” C. Expired air is passed through a carbon dioxide meter to measure partial pressure of carbon dioxide. Eucapnic conditions are maintained by adding carbon dioxide to the inspired gas at a rate sufficient to maintain end tidal partial pressures between 38 and 42 mm Hg. The procedure may be simplified by having patients inspire dry gas at room temperature, thus obviating the need for a heat exchanger.3’ At room temperature hyperventilation of dry gas at ventilation levels comparable to those achieved during strenuous exercise (60 to 70 L/min) can trigger airway narrowing in most patients with asthma. Cooling of the inspired air can potentiate the response. The optimum duration of hyperventilation is between 3 to 4 minutes. By varying the level of the stimulus intensity, one can use eucapnic hyperventilation to obtain graded stimulus response curves. This provides a means of evaluating the effects of a drug on the response to increasing levels of stimulus intensity, thus providing more complete information on the degree of protection. The use of eucapnic hyperventilation challenges to obtain stimulus response curves was based on earlier observations that hyperpnea failed to induce a refractory period similar to exercise. More recent studies, however, have demonstrated refractoriness after hyperpnea. It seems clear, however, that graded stimulus response curves can be obtained with eucapnic hyperventilation despite the occurrence of refractoriness, and that the reproducibility of such curves is adequate for drug testing. The most common practice is to vary the stimulus intensity by changing the minute ventilation level at fixed inspired air conditions. Varying levels of ventilation in onefold or twofold increments, starting at 7.5 L/mm and increasing to maximum voluntary ventilation, can be performed in
The role of bronchoprovocation
521
sequence, allowing for a brief interval between challenges for measurement of the response. The response is plotted against the stimulus intensity. The stimulus intensity is most simply expressed as the minute ventilation provided that inspired air conditions are kept constant at different levels of hyperpnea. Alternatively, the stimulus can be expressed in terms of units of respiratory heat exchange. However, the use of these units as an expression of stimulus intensity is somewhat controversial because of either unproved assumptions concerning expired air conditions or technical difficulties in actual measurements of expired air temperature. Calculations of respiratory heat exchange in many early studies were often based on assumed values of expired air temperature and humidity rather than actual measurements. The assumption that expired air is completely saturated under all temperature conditions has been questioned, and further experiments are needed to confirm this assumption. Furthermore, because of the limited response times and calibration problems of most commercially available temperature recording devices, the precision of expired air temperature measurements has been questioned. At present, minute ventilation is the preferred expression of the stimulus intensity. Units of respiratory heat exchange are also considered acceptable, provided that the investigators acknowledge whether expired air temperature and humidity are in fact measured or whether assumed values are used. The airways response to the graded stimulus is measured before drug administration, after administration just before the test, and after the test at l-minute and 2 to 3-minute intervals until the peak of the response is obtained. Allergens The principles for regulation of allergen inhalation tests are similar to those for inhalation tests with chemical mediators.25.26Differences result from problems with standardization of test extracts, the dependence of the early asthmatic response on the level of IgE antibodies, airway responsiveness to endogenous release of chemical mediators and the dose of allergen, the different time course of the early asthmatic response that peaks later, and the frequent occurrence of late asthmatic responses with an associated prolonged increase in airway responsiveness. Crude allergen extracts are not well characterized with respect to the number and concentration of antigens they contain. Purified allergens, such as antigen E derived from crude ragweed extract, are available for some exogenous causes of asthma. In any case, there must be enough of the same extract lot for each
522
Hargreave
et al.
subject in the study. Furthermore, crude or purified allergen extracts degrade with time. The degradation can be reduced by storage between 4” and - 20” C and by the addition of a stabilizer such as human serum albumin. When the effect of a drug on the allergeninduced response is examined, allergen extracts should be stored at - 20” C in freeze-dried form or as the concentrate in single-use aliquots. The extracts should be reconstituted or thawed and diluted shortly before use on the same day. They should not activate complement nonspecifically. The allergen-induced early asthmatic response is chiefly determined by the level of specific IgE antibodies, the dose of antigen, and the level of airway responsiveness to histamine or methacholine.23 The first two factors determine the presence and degree of mediator release and the latter indicates the effect of the mediators on airway structures. At the start of the study, threshold allergy skin tests are required to determine a safe dose to begin allergen inhalations.23 The allergen-induced early asthmatic response is usually advanced at 10 minutes and peaks between 10 and 30 minutes. As a result, measurements of the response are conveniently made at about 10 minutes. Allergen inhalations are stepped up until there is a response equivalent in severity to a fall in FEV, of about 15% to 20%. Convenient times to follow the early response are 5, 10, 15, 20, and 30 minutes and 1, 1.5, 2, and 3 hours. When repeat tests are made, the starting dose of allergen should be at least two twofold doses below the final dose in the first test. Late asthmatic responses commonly occur after allergen inhalation and must be recorded by the repetition of measurements each hour for at least 8 hours after the allergen-induced early response. Their severity cannot yet be predicted, although three important determinants have been indicated: ( 1) the dose of allergen, (2) the severity of the early asthmatic response, and (3) the baseline histamine or methacholine airway responsiveness. Their association with prolonged increases in histamine or methacholine airway responsiveness may make it impossible to repeat allergen inhalation tests until the hyperresponsive state has returned to baseline (for 1 or more weeks).
SAFETY The primary consideration in the use of provocation test protocols must be the protection of the health and safety of the subjects. Because of the potential risks associated with the challenge procedure itself, studies should be carried out only in patients who have been appropriately screened and found suitable on the basis of clinical status and pulmonary function stability. Moreover, because of the potential for severe and un-
J ALLERGY
CLIN. IMMUNOL. SEPTEMBER 1986
expected reactions to allergens, it is recommended that allergen inhalation testing be performed only by investigators experienced in these procedures in a setting in which untoward reactions can be anticipated and properly managed. When these tests are used in the evaluation of drug efficacy, proper informed consent for challenge protocols must be reviewed and approved by institutional review boards.
EXAMINATION OF EFFECTS OF DRUGS The same scientific principles that apply to the study design and analysis of all drug trials, specifically regarding definition of the study question, treatment controls, blinding, randomization, and so forth, must also be applied to provocation test studies. A number of additional points particularly relevant to provocation tests are discussed here. The reproducibility of responses in each laboratory should be known and should match the expected reproducibility. Reproducibility can vary between individuals and studies and therefore, if there is doubt, it should be assessed in the study design. The response to the test can be altered by the previous test, and therefore consideration should be given to the interval between tests to try to avoid this possibility. For example, the response to exercise can be followed by a refractory period for 2 to 3 hours during which the response to another exercise test will be reduced. It is therefore recommended that exercise tests be repeated at intervals of 3 to 4 hours or more to avoid this possibility. The best method to examine the magnitude of effect of a drug on early asthmatic responses is to measure the change in position of the stimulus response curve. This is possible in all provocation tests with the exception of exercise, for which stimulus response curve determinations take too long to be practical. When allergen-induced early responses are examined in this way, there is the possibility of causing a subsequent severe late asthmatic response if the drug inhibits the former response more than the latter. Therefore, the patient must be carefully followed for this possibility or treated to prevent it. It is also recommended that a time course study be performed to determine the optimum time zt which a drug might be expected to exert its effect after the allergen challenge. For example, an H,-receptor antihistamine should be evaluated within 5 minutes after the challenge, because histamine plays a major role in immediate bronchoconstriction. On the other hand, the optimal time to examine an anti-LTD, effect is between 10 and 30 minutes after challenge, because membrane-generated mediators account for most of the bronchoconstrictor effect at this time.
VOLUME NUMBER
The role of bronchoprovocation
78 3. PART 2
Examination of the effect of drugs on allergen-induced early and late asthmatic responses and increases in airway responsiveness can be important to examine, but the best approach to the study design needs investigation. However, at present there are too many uncertainties about the variables that influence results to be able to draw up guidelines on a factual basis. Nevertheless, it is felt that these gaps in our understanding should not be a deterrent to studies of drug effects on allergen-induced responses. Rather, carefully designed protocols to investigate drug effects in this model of acute airway inflammation are encouraged as a means of advancing our understanding of drug effects as well as the disease we seek to treat. Baseline airway caliber can influence results. This is less likely in subjects with normal baseline measurements and in short-term studies with NBAAD. In long-term studies, however, these drugs can produce bronchodilation as a secondary event. At present, it is not known how to analyze the effect of a change in airway caliber, but absolute predrug and postdrug values, predicted values, and percentage changes should all be reported (see workshop 5). One method recently used to investigate the effect of change in airway caliber has been to adjust for the change by analysis of covariance.32 The analysis of results of PD, or SGaw, should be carried out on log-transformed data. Almost all statistical procedures require that the inherent variability of an outcome measure be constant over subjects or groups of subjects. This is not true for PD,, or SGaw,. for which individual values from different patients may differ by two logs or more. A logarithmic transformation (either natural or to base 10) of PDZO and SGaw, eliminates the tendency for standard deviation to be related to the mean. All statistical comparisons should thus be conducted after first performing log transformation of individual values. Summary statistics can be quoted either on the transformed scale or after conversion to a linear scale. Although the logarithmic transformation is used here primarily to stabilize variance, it will also tend to produce a more normal distribution and perhaps linearize relationships with other factors such as drug dose. SUMMARY Provocation tests can be. used to investigate the effects of NBAAD. These studies provide scientific information on the pharmacology of drugs in humans, their potential clinical efficacy, and the optimum dose and duration of their effect. They serve as a useful link between preclinical studies and clinical trials; however, they do not necessarily identify the clinical value of drugs and they are not a substitute for clinical
523
trials. Provocation tests include inhalation tests with chemical mediators of airway narrowing, tests with natural nonsensitizing stimuli such as exercise and hyperventilation, and inhalation tests with allergens. Each of these stimuli acts through different specific mechanisms. All of them can trigger early asthmatic responses; exercise can also stimulate late asthmatic responses; and allergens can induce late asthmatic responses and prolonged increases in airway responsiveness to chemical mediators. The provocation tests must be carefully regulated to produce reproducible responses against which the effect of the drugs can be examined. This stipulation requires that the patient, environment, stimulus, and airways measurements are the same for each treatment condition. The study design must incorporate the scientific principles applicable to all drug studies. While guidelines can be given to studies that involve tests with chemical mediators, exercise, and hyperventilation, they cannot yet be specified for allergen inhalation tests, for which the factors that influence reproducibility are less well understood. REFERENCES 1. Pepys J, Chan M, Hargreave FE, McCarthy DS. Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 1968;2: 134-7. 2. Booij-Noord H, Orie NGM, de Vries K. Immediate and late bronchial obstructive reactions to inhalation of house dust and protective effects of disodium cromoglycate and prednisolone. J ALLERGY CLIN IMMUE~OL1971;48:344-54. 3. Phillips MJ, Oilier S, Gould CM, Davies RJ. Effect of antihistamines and antiallergic drugs on responses to allergen and histamine provocation tests in asthma. Thorax 1984;39:34551. 4. Juniper EF, Latimer KM, Morris MM, Roberts RS, Hargreave FE. Airway responses to hyperventilation of cold dry air: duration of protection by cromolyn sodium. J ALLERGY CLIN IMMUNOL 1986;78:387-!)l. 5. Godfrey S, Konig P. Inhibition of exercise-induced asthma by different pharmacologic pathways. Thorax 1976;3 1: 137-43. 6. Barnes PJ, Wilson NM, Vickers H. Prazosin, an alpha,adrenoceptor antagonist, partially inhibits exercise-induced asthma. J ALLERGY CLIN IMMUNOL 198 1;68:4 11-5. 7. Conis PA, Nariman S, Gibson GJ. Nifedipine in the prevention of asthma induced by exercise and histamine. Am Rev Respir Dis 1983;128:991-2. 8. Fairfax AJ, Hanson JM, Morley I. The late reaction following bronchial provocation with house dust mite allergen. Dependence on arachidonic acid metabolism. Clin Exp Immunol 1983;52:393-8. 9. Joubert JR, Shephard E, Mouton W, Van Zyl L, Viljoen I. Non-steroid anti-inflammatory drugs in asthma: dangerous or useful therapy? Allergy 1985;40:202-7. 10. Hargreave FE, Sterk PJ, Ramsdale EH, Dolovich J, Zamel N. Inhalation challenge tests and airway responsiveness in man. Chest 1985;87:S202-6. 11. Hargreave FE, Woolcock AJ. Airway responsiveness: measurement and interpretation. Mississauga: Astra Pharmaceuticals Canada Ltd. 1985.
524
Hargreave
J. ALLERGY
et al.
12. Hardy CC, Robinson S, Tatterfield AE, Holgate ST. The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men. N Engl J Med 1984;311:209-13. 13. Thomson NC, Roberts R, Bandouvakis J, Newball H, Hargreave FE. Comparison of bronchial responses to prostaglandin F, and methacholine. J ALLERGY CLIN IMMUNOL 1981;68: 392-8. 14. Adelroth E, Morris MM, Hargreave FE, O’Byme PM. Airway responsiveness and relative potency of leukohienes C,, D, and methacholine in asthmatics and normals. N Engl J Med (in press). 15. Griffin M, Weiss SW, Leitch AG, et al. Effects of leukotriene D on the airways in athma. N Engl J Med 1983;308:436-9. 16. Cushley MJ, Church MK, Pao GJ-K, Holgate ST. The role of mast cell mediator release in adenosine-induced bronchoconstriction. J ALLERGYCLIN IMMUNOL 1985;75:272-8. 17. Cartier A, Malo JL, Begin P, Sestier M, Martin RR. Time course of the bronchoconstriction induced by inhaled histamine and methacholine. J Appl Physiol 1983;54:821-6. 18. Ruffin RE, Alpers JH, Crockett AJ, Hamilton R. Repeated histamine inhalation tests in asthmatic patients. J ALLERGY CLIN IMMUNOL 1981;64:285-9. 19. Ryan G, Latimer KM, Juniper EF, Roberts RS, Hargreave FE. Effect of beclomethasone dipropionate on bronchial responsiveness to histamine in controlled nonsteroid-dependent asthma. J ALLERGY CLIN IMMUNOL 1985;75:25-30. 20. Lowhagen 0, Rak S. Modification of bronchial hyperreactivity after treatment with sodium cromoglycate during pollen season. J ALLERGY CLIN IMMUNOL 1985;75:460-7. 21. Sotomayer H, Badier M, Vervloet D, Orehek J. Seasonal increase in carbachol airway responsiveness in patients allergic to grass pollen: reversal by corticosteroids. Am Rev Respir Dis 1984;130:56-8. 22. O’Byme PM, Morris M, Roberts R, Hargreave FE. Inhibition
23.
24.
25. 26.
27.
28. 29.
30. 3 1. 32.
CLIN. IMMUNOL. SEPTEMBER 1986
of the bronchial response to respiratory heat exchange by increasing doses of terbutaline sulphate. Thorax 1982;37:913-7. Hargreave FE, Frith PA, Dolovich M, et al. Allergen-induced airway responses and relationships with nonspecific reactivity. In: Hargreave FE, ed. Airway reactivity: mechanisms and clinical relevance. Mississauga: Astra Pharmaceuticals Canada Ltd, 1980:145-50. Hargreave FE, Dolovich J. Bronchial responsiveness and late asthmatic response. In: Kay AB, Lichtenstein LM, Austen KF, eds. Asthma: physiology, immunopharmacology and treatment. London: Academic Press, 1984:263-77. Hargreave FE, Dolovich J. Boulet L-P. Inhalation provocation tests. Semin Respir Med 1983;4:224-35. Davies RJ, Corrado OJ. Diagnostic tests-challenge tests: oral, nasal and bronchial. In: Lessof MH, ed. Allergy, immunological and clinical aspects. Chichester: John Wiley & Sons, 198483-105. Fish JE, Kelly JF. Measurements of responsiveness in bronchoprovocation testing. J ALLERGY CLIN IMMUNOL 1979;64: 592-6. Godfrey S. Childhood asthma. In: Clark TJH, Godfrey S, eds. Asthma. 2nd ed. London: Chapman and Hall, 1983:415-56. Laube BL, Adams GK, Norman PS, Rosenthal RR. The effect of inspiratory flow rate regulation on nebulizer output and on human airway response to methacholine aerosol. J ALLERGY CLIN IMM~JNOL1985;76:708-13. Scott GC, Kung M. How many spirograms for a histamine challenge? Am Rev Respir Dis 1985;132:268-71. Rosenthal RR. Simplified eucapnic voluntary hyperventilation challenge. J ALLERGYCLM IMMUNOL 1984;73:676-9. O’Bryne PM, Thomson NC, Morris M, Roberts RS, Daniel EE, Hargreave FE. The protective effect of inhaled chlorpheniramine and atropine on bronchoconstriction stimulated by airway cooling. Am Rev Respir Dis 1983;128:611-7.