Use of provocation techniques for the evaluation of drug efficacy

Use of provocation techniques evaluation of drug efficacy Sheldon

L. Spector,

M.D. Denver, Co10

The chairman of this round-table discussion, Dr. David Herzig, asked me to address the following three questions. I will respond to them in this order: (1) How does one select patients for bronchial challenge in terms of (a) disease, (b) present therapy, and (c) sample size? (2) What baseline and placebo studies are needed? (3) Does blocking of bronchial challenge with a drug predict actual efficacy of the drug in a double-blind clinical investigation? SELECTION OF PATlENT BY DISEASE, FOR BRONCHIAL INHALATION CHALLENGE Asthmatic patients, by definition, have reversible obstructive airway disease. Such patients need not be allergic and need not wheeze. There are patients who have asthma without wheezing’ or have a chronic cough as a manifestation of airway obstruction. Often what the chest physician designates as chronic obstructive pulmonary disease, the allergist designates as reversible obstructive airway disease (ROAD). There is controversy as to the extent of reversibility necessary for the designation, ROAD. In the literature, patients have been categorized according to their predicted values as well as their best pulmonary function values during a certain observed period of time. It should be remembered that predicted value is not a single number but a range emanating from a mean value. Although I prefer to look at asthmatic patients in terms of various degrees of reversibility, theoretically, results might differ depending on how reversibility is defined. An example of an alternative idea to the one which necessitates that the patient’s pulmonary functions fall within predicted values is to exclude patients with a I-set forced expiratory volume (FEV,) of less than 1 1. Such a consideration mitigates against small absolute changes in pulmonary function being judged as a positive response. Other respiratory disorders should exclude a patient

From the Department of Medicine, National Jewish Hospital and Research Center. Reprint requests to: Sheldon L. Spector, M.D., National Jewish Hospital and Research Center, 3800 E. Colfax Ave., Denver, CO 80206. 0091-6749/79/l

30677+08$00.80/O

for the

@I 1979 The C. V. Mosby

from entering into a study. Needless to say, a patient might wheeze from cardiac problems. There are other reasons for excluding certain patients such as a concomitant serious illness. Reactivity to methacholine and histamine should also be part of the definition. Although there are some patients with negative methacholine and histamine challenges who resemble asthmatics in almost every other way, these patients are quite rare. Thus, an increased reactivity to methacholine and histamine has even been suggested as an integral part of the definition of asthma.‘, 3 If an investigator wishes to test the blocking effect of an agent on bronchial inhalation challenges, the patient selection criteria should include only those patients with a known endpoint response that occurs at least 2 dilutions before administration of the maximum concentration. Needless to say, the patients should be age and sex matched and randomized as part of an appropriate design. Ideally, smokers should be handled separately from nonsmokers and individuals with reagin-mediated disease separately from non- reagin-mediated disease. There are special considerations one should keep in mind depending on the agent to be tested. For example, if a new oral cromolyn agent is to be assessed, it would make sense to test that agent for effectiveness after the patient’s usual cromolyn has been discontinued. SELECTION OF PATIENTS PRESENT THERAPY

BY

There are few, if any, controlled studies that have assessed the most appropriate length of time that medications should be withheld in order to avoid their influence on bronchial inhalation challenges. Ideally, all medications should be discontinued for all patients. However, this is impractical in most situations, since the numbers of patients one could thereby test would be so small as to mitigate against an adequate sample. Next best would be patients receiving a minimal number of drugs without corticosteroids, and least desirable for study are patients on many medications as well as corticosteroids. On the other hand, some studies may be designed to assess a more difficult patient population such as this latter group. In Co.

Vol. 64, No. 6, Part 2, pp. 677-684

678

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J ALLERGY

Aerosolized Methacholine Response 40

f

g

20

2 0

10

Y

-1 5

2

2

I

T40

-L

-

Control

1 P YPp’i I II P

"w""Ir-Jnaa~+g momoaumumaSZx

Marked Responders

FIG. 1. Change in threshold response to aerosolized methacholine with troleandomycin (TAO): ., prior to TAO; O, during TAO. As indicated, a typical asthmatic patient not receiving TAO may have a variance of 1 breath sequence when tested on 2 separate occasions. Patient S. G. had a negative response to 40 breaths of 2.5 mg/cm3 but his response to 10 mg/cm3 was not tested.

any case, medication should be withheld if there is potential for interfering with proper assessment of the results of the experiment or at least held as constant as possible on control days compared to experimental days. Patients receiving daily steroids should be included in a study only if they will continue their daily steroids throughout the study. Patients receiving alternate-day corticosteroids might best be tested on their “off corticosteroid” day. At least there should be consistency. Corticosteroid aerosols have potential for blocking late reaction? so it would be inappropriate to continue these medications if an investigator is specifically looking for changes in thresholds of early and late responses to antigen inhalation challenge. Although cromolyn sodium should probably be withheld at least 24 hr, as mentioned previously, it would be inappropriate to continue medication for the patient if the full potential of an oral cromolyn agent is to be tested in that patient. Recommendations to withhold antihistamines for 48 to 96 hr are extrapolated from skin test data.” Although beta adrenergic agents should be withheld for 8 hr, another important consideration is that there be a

CLIN. IMMUNOL DECEMBER 1979

2-wk washout period prior to starting a new adrenergic agent. Phosphodiesterase inhibitors are withheld according to what type of preparation is used. Shortacting medications should be withheld at least 6 hr and long-acting theophylline preparations for 12 hr. A more strict criterion could be established with the use of theophylline blood levels. Other medications may also contribute toward interfering with appropriate assessment of a bronchial inhalation challenge result in rather unexpected ways. Thus, an antibiotic such as troleandomycin might have a profound effect on methacholine challenge reactivity, as shown in Fig. 1. SELECTION OF PATIENTS SAMPLE SIZE

ACCORDING

TO

Although a discussion such as this might most appropriately be handled by a statistician, I shall make certain generalizations derived largely from protocols using bronchoconstrictive and bronchodilator agents. General

considerations

1. Talk to the statistician ahead of time regardmg the design of the protocol. 2. Decide how the data will be analyzed in advance, including a section on statistical method. 3. Graph the data, looking for trends or bimodal distribution. Computers are capable of graphing and can save a lot of time. 4. Use placebos and washout periods when applicable. 5. Randomize patients. A strict randomization requires a larger sample size than one based on a Latin square design, which also takes the “order effect” into consideration. 6. It is much easier to prove there is a difference than to prove no difference. 7. With enough data, a statistician will find a difference. However, it may have no clinical significance. The clinician ultimately must make a decision. 8. The t test (2 sample) is probably the most abused test in the literature. It should not be used to compare more than 2 groups and it is only valid if a single attempt at comparison is made. It is not legitimate to compare 1 group to another 6 times or 100 times, since the odds change proportionately. The t test also assumes a normal distribution so if there is not a normal distribution, a nonparametric test should be used. Specific

considerations

With the aforementioned considerations in mind, the sample size will increase as the number of drugs studied and the number of doses of the drug increase.

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64 6, PART 2

TABLE I. P values

Evaluation

for various

sample

of drug efficacy

679

sizes P value

Patients showing improvement (%) so 55 60 65 70 75 80 x5 90 95 100

Sample size 3

Sample size 5

Sample size 15

0.6230 0.5000

0.3770

0.5000

0.5000 0.3036 0.1509

0.1719 0.1875

0.0547

0.0592 0.0176 0.0037

0.0107 0.1250

0.03 13

TABLE II. Effect of fenoterol, aminophylline, reactivity with a variety of allergens Drug

Sample size 10

0.0010

and placebo

Wheel

0.0005 0.0000

Sample size 20 0.588 I 0.41 I9 0.2517 0.1316 0.5777 0.0207 0.0059 0.0013 0.0002 0.0000 0.0000

on skin test

Baseline*

1 hr. 50 min. after drug*

Differencet

Placebo

Diameter (mm) Area (mm’)

7.8 71.6

7.2 68.7

0.6 2.9

Fenoterol

Diameter (mm) Area (mm’)

7.6 88.0

5.8 65.6

I.8 22.4

Aminophylline

Diameter (mm) Area (mm’)

8.6 81.7

7.6 80.3

I.0 I .4

*Expressed as mean value minus the diiuent control value. TDifference represents the mean of the largest diameter or area at baseline minus the I hr. 50 min, value. All values were not significant, p
The number of measurements made per patient per drug also influences the total sample. Statistically, it is easier if the patient serves as his own control as opposed to comparing him to different sets of people. If 5 people are studied in detail, the conclusions are on firmer statistical ground than if different sets of people are studied, but extrapolation to the general population is more difficult. The size of the difference to be detected also influences sample size. Thus, if the clinician looks for a small difference in highly variable data, he will need a large number of patients to get a significant result. Lack of statistical significance may indicate that alone or it can be due to the sample size. The variability of the measurements on patients also influences sample size. If subject-to-subject variation is considerable, the clinician must study more patients to obtain statistically meaningful’ results. A useful tool employed to estimate numbers is shown in Table I. First, let us assume that 1 dilution, or perhaps 1 log, is the variability expected from doing 2 controlled challenges, and that 2 or more orders of magnitude of change in the right direction

would indicate the blocking effect. As seen in Table I, if the clinician decides that 75% of his patients must show improvement before this becomes a clinically important observation, approximately 20 patients would be needed for a p value of 0.05. If the investigator is not familiar with many of the variables, sometimes a legitimate technique that can be utilized is to finish complete testing of 5 patients, and to send the data to the statistician, who can analyze it. The statistician can determine the appropriate sample size without breaking the code to the investigator. NECESSARY BASELINE AND PLACEBO STUDIES Baseline values for pulmonary function studies should be done with and without diluent. The diluent baseline is then used as the basis for comparison of the test substance. Since suggestion may influence pulmonary function tests, especially airway resistance and specific conductance, protocols must be designed with this consideration in mind.fi Two practical approaches to

680

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J ALLERGY

Aminophylline

8

6

Placebo f

.1 -2 74 C

fj -4

’

-6 -6

FIG. 2. Blocking effect of placebo and aminophylline during methacholine inhalation challenge in an individual patient (comparisons of initial values with medication values, expressed by changes in standardized concentration increments).

deal with the effect of suggestion would be (1) doing the bronchial inhalation challenges in a setting where neutral substances, bronchodilators, and bronchoconstrictors are all given to the patient so that he does not know what to expect and (2) not telling the patient which of the 3 possibilities he might expect until after the experiment is complete. The baseline initial values may influence subsequent response. Hume and Gandevia7 reported that as the FEV and forced vital capacity (FVC) improve, so does the absolute response, until it reaches a maximum. They found that the absolute response was poor if the initial value was low. Goldberg and CherniackX described the greatest changes after therapy as taking place when airway resistance was highest. Initial values may in fact reflect pathogenic mechanisms, whereby at a given time there may mainly be mucosal edema in 1 patient compared to bronchospasm in another. The investigator must choose the pulmonary function measurements. Ideally, one would want both spirometry and body plethysmography to determine volume changes; however, such sophisticated equipment is not always available. By contrast, the peak flow measurements alone can prove useful in certain studies where the patient serves as his own control and performs the maneuvers frequently during the day. Such values are better than no data at all, if the patient is properly instructed and is reliable. Reliability can often be assessed by obtaining a simultaneous curve, such as with flow volume loops. The number of measurements and timing also

CLIN IMMUNOL DECEMBER 1979

should be stipulated. The best of 3 values is preferred: however, fatigue can ensue if the patient performs too often within a short period of time. Since the spirometric maneuver itself can cause bronchoconstriction,” panting should precede spirometry. The effect of fatigue itself should be considered if there is a long series of dilution sequences and an unclear endpoint. This was one reason the standardization panel considered 5 breaths rather than 10.” The position for pulmonary function maneuvers assumed by the patient is also important. Pierson et al. ‘I found, in a study of 235 subjects, that sitting values were slightly higher than standing values if FVC was greater than 2.14 1, FEV, was greater than I .68 1. FEFzs-m* was greater than 2.16 1, and FEV, /FVC was greater than 75.7%. Subjects with values falling below the aforementioned performed slightly better in the standing position. The time of day also influences results, probably due to various circadian and body rhythms. Gervais et al.‘” found a 24-hr rhythm in bronchial hyperreactivity to house dust in asthmatics. At 8:00 A.M. there was the least effect and at 11:OO P.M., the greatest effect in terms of persistence of bronchospasm after challenge. Bronchial reactivity to histamine and acetylcholine also follows certain rhythms. No upper respiratory infections should have been present and no vaccines should be administered for weeks prior to initiation of the study; results of challenges to methacholine or other chemical substances are known to be influenced.2 The frequency with which the tests can be repeated varies somewhat depending on the challenge to be used. The effects of bronchial challenges with methacholine and antigens seem to be cumulative while those with histamine are not. Thus, it may make sense to express data in a cumulative fashion for the former 2 but not for the latter. In general, it would be appropriate to avoid more than 1 challenge on the same day. DOES BLOCKING BRONCHIAL CHALLENGE WITH DRUG PREDICT EFFICACY OF SAME IN DOUBLE-BLIND CLINICAL INVESTIGATION? What are alternatives to bronchial challenges in man? Drug manufacturers customarily use various in vitro techniques and animal models to test a new medication. Limitation of in vitro, models is well known. A good example is the important limitation of the use of in vitro measurement of sensitivity of peripheral leukocytes (basophils) as an index of a per*Mean rate of expiratory air flow between 25% and 75% of forced expiratory vital capacity.

VOLUME NUMBER

TABLE III. Discharge Dr.

Evaluation of drug efficacy

64 6. PART 2

681

diagnoses

_

No Yes ? Eczema

I29

Rhinitis

I29 I

Reagin-mediated

2

9

Nasal polyps

129

Sinusitis (by x-ray)

I

2

9

Chronic bronchitis (Am. Thor. Sot. definition)

1

2

9

Yes 2 2

? 9 9

Asthma I. infections I 2 3 4 A. Infrequent B. Not severe 1 2 3 4 2. Reagin-mediated A. By skin test B. By bronchial challenge 3, Hyperreactive airways A. Admissions testing I. To histamine

5 5

Details

or comments

Frequent Severe No 1 I

High concentration

Low concentration

2. To methacholine B. Discharge testing I. To histamine

0.03

0.06

0.12

0.08

0.15

0.31

0.03

0.06

0.12

0.25 0.50 or Negative result or Not 0.62 1.25 or Negative result or Not 0.25

0.50

1.00 2.50 tested 2.50 5.00 tested 1.00

(mglml)

5.00

10.00

10.00

25.00

2.50

5.00

10.00

5.00

10.00

25.00

or Negative result or Not tested

2. To methacholine

0.08

0.15

0.31

0.62

1.25

2.50

or Negative result or Not tested

4. Reversibility None A. After aerosolized I bronchodilators B. After medications (including 1 corticosteroids) 5. 6. 7. X. 9. IO.

Complete

2

3

4

5

2

3

4

5

No I I I I I 129

Yes 2 2 2 2 2

? 9 9 9 9 9

Exacerbation by exercise Exacerbation with menstruation Aspirin idiosyncrasies by challenge Tartrazine intolerance by challenge Acetaminophen intolerance Sodium salicylate intolerance

12. Medications A. Alpha adrenergic blockers B. Beta adrenergic agonists I. List medications and a. b. C.

d. e. 2. Idiosyncratic response

Response during hospitalization *

01239

route, and evaluate 01239 01239 01239 01239 01239 Yes ? No 1 2 9

*O, Not given; 1, better; 2, same; 3, worse; 9, ?.

Class:

1

11

Prescribed No

12

12 12 12 I2 I2

Yes

at dischurge

DosagelComments

111

IV

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J ALLERGY

CLIN. IMMUNOL DECEMBER 1979

TABLE Ill. cont’d 12. Medications-cant ‘d C. Aminophylline and other phosphodiesterase inhibitors I. 0 I 2 3 9 2 I I 2. 0 I 2 3 9 2 D. Investigational drugs 2 I I. 0 I 2 3 9 2 I 2. 0 I 2 3 9 E. Anticholinergic agents (e.g., atropine) List medications and route, and evaluate 2 I I. 0 I 2 3 9 I 2 2. 0 I 2 3 9 F. Cromolyn sodium (and 0 I 2 3 9 I 2 analogs) G. Corticosteroids (including inhaled) List medications and route, and evaluate 2 I I. 0 I 2 3 9 2 2. 0 I 2 3 9 I 2 I 3. 0 I 2 3 9 H. Antihistamines List medications and route, and evaluate 2 I I. 0 I 2 3 9 2 2. 0 I 2 3 9 I 2 3. 0 I 2 3 Y I 2 I. Troleandomycin 0 I 2 3 9 I 2 J. Iloxone 0 I 2 3 9 I 2 I K. Potassium iodide 0 1 2 3 9 L. List other medications and routes, and evaluate 2 I. 01239 I 2 2. 01239 1 2 3. 01239 I 2 4. 01239 I 2 5. 01239 I Suituble 7 Unsuitable 13. Reliability index 3 I 2 Is this patient suitable for research study? 14. Additional diagnoses:

son’s hypersensitivity, which is a means of monitoring efficacy of injection therapy. As mentioned previ-

ously, May and Williams’” could render blood basophils unresponsive to antigen challenge, yet patients kept their skin reactivity, their immunoglobulin E levels, and their clinical sensitivity. Exposure of isolated mast cells to antigen also means that the autonomic nervous system and higher neurologic pathways responsible for suggestion, etc.,

Comments

are dissociated from the response. Yet their roles in physiologic processes are very important. Animal models also have certain limitations. The greater the phylogenic difference between animals and man, the greater the chance that the animal model will not be applicable to man. The characteristics of certain animal models must be ascertained prior to performing the experiment in order for a meaningful extrapolation to be made to the human being. Thus,

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Evaluation

64 6, PART 2

the rabbit, mouse, and rat are steroid-sensitive animals, while the guinea pig or monkey (like man) are steroid resistant. I’ In rats and mice, serotonin is one of the important mediators released from mast cells, but not in man.‘: Also, experiments done in monkeys and primate models may differ from subsequent experiments done in man himself. Miller and Patterson’” failed to demonstrate bronchial hyperreactivity to physostigmine in rhesus monkeys with reagin-mediated asthma, even though Miller et a1.17were able to demonstrate bronchial hyperreactivity to physostigmine in asthmatic subjects. Perper et al. Ix could inhibit the Ascuris skin reaction with the selective beta-2 agent, salbutamol (albuterol). yet they and other investigators could not demonstrate a blocking effect with beta adrenergic stimulators during allergenic skin tests in man.‘!‘-” Table II shows my data for fenoterol; neither this selective beta agonist nor theophylline inhibit antigen skin test reactivity. Sometimes, it is not the animal model itself that is responsible for the discrepancy but rather the techniques and doses of medication used that explain the different results. In some circumstances, there are no alternatives to in vitro or animal models. Some experiments cannot be done ethically in human beings. Those of us who do clinical studies know only too well that it takes a lot of time and energy to properly perform human experimentation. The informed consent of the patient and the various ethics committees helps ensure propriety. Human studies must be designed carefully to best assess the effect on bronchial inhalation challenge. Patient selection may be the most important factor for proving clinically meaningful associations, as has been discussed previously. For example, a truly reagin-mediated population might be most appropriate to demonstrate effectiveness of a cromolyn-like substance and a young population might be best to assess the effect of a new agent on exercise-induced bronchospasm. Ideally, the organ of involvement should be tested. Thus, the lungs should preferentially be tested in asthmatics rather than the skin. Other organs also might be tested for a different purposefor example, to determine whether a certain medication should be withheld during skin testing procedures. The medication should be tested as directly as possible for the use desired. If the main indication of a new drug is bronchodilatation without concomitant cardiovascular effect, pulmonary function measurements should be assessed along with cardiovascular parameters. Too many people erroneously equute ul-

of drug efficacy

683

nlith asrhmu, yet the most common characteristic of an asthmatic is the hyperirritability of his bronchial tree rather than bronchospasm secondary to reagin. With these considerations in mind, methacholine challenges might serve as a better model for asthma than antigen inhalation challenges. Many studies have shown that both beta adrenergic agents and anticholinergic agents block histamine or methacholine challenges. On the other hand, atropine probably does not block antigen challenge,“’ yet atropine has shown itself to be a useful clinical agent. Finnish authors have described how ICI 74,917, a phenanthrolene derivative, diminishes allergen-induced bronchoconstriction but fails to clinically improve asthma symptoms compared to placebo after 1 mo of treatment.““i Should one expect to make meaningful statements regarding clinical efficacy from the results of an antigen inhalation challenge‘? These same authors used concentrated solutions of antigens, which, in themselves, might not be physiologic, thus making clinical judgments that much more difficult. It is imperative that designs be clinically applicable through utilization of standardized techniques. If the “egg law” is broken due to high concentrations of antigens, the specificity or clinical usefulness of the test, as has been mentioned previously, may be questionable. The dose of the medication can be critical and dose-response assessments are ideal. A blocking drug should also be administered so that it peaks at an appropriate time in relation to the bronchial inhalation challenge; thus, timing is important. Statistics often mask individual responders. Isolated patients who seem to be remarkable responders should be cherished and retained for future studies that have better sorted out the heterogeneity of the patient population. A good example of such reasoning is phentolamine, an alpha adrenergic blocker, which was found to be very useful in a particular patient in whom the usual bronchodilators failed. My group has subsequently challenged many patients and found the remarkable bronchodilatation in only 1 or 2 more. Another example is shown in Fig. 2. This is an individual in whom aminophylline markedly blocked methacholine (and histamine) challenges. Yet in another study we could not show statistically that aminophylline significantly blocked these challenges.‘A Perhaps our ultimate clinical goal should be to tailor-make a program based on individuals’ evaluated responses. We have attempted to do this with our discharge diagnoses shown in Table III. Although such a consideration may be impractical, it has the most meaning to an individual patient. In summary, bronchoprovocation tests certainly

lcrgy

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Spector

have potential to better predict efficacy of a drug than in vitro models or animal models. With the right patient selection and the proper standardization, clinical usefulness might be confirmed statistically; the ability of a drug to block bronchial challenges also has potential clinical usefulness on an individual basis. REFERENCES I. Farr RS. Kopetzky MT, Spector SL, Hurewitz DS: Asthma without wheezing. Chest 63:643, 1973. 2. Spector SL. Farr RS: A comparison of methacholine and histamine inhalation in asthmatics. J ALLERGY CLIN IMMUNOI 65:308, 1975. 3. Spector SL, Farr RS: Bronchial provocation tests, in Weiss EB. Segal MS, editors: Bronchial Asthma Mechanisms and Therapeutics. Boston. 1976, Little, Brown & Company, pp. 639-647. 4. Pepys J. Davies RJ. Breslin ABX, Hendrick DJ, Hutchcroft BJ: The effects of inhaled beclomethasone dipropionate (Becotide) and sodium cromoglycate on asthmatic reactions to provocarion tests. Clin Allergy 4: 13, 1974. 5. Galant SP, Bullock J, Wong D, Maibach HI: The inhibitory effect of antiallergy drugs on allergen and histamine induced wheal and flare response. J ALLERGY CLIN IMMUNOL 51: I I, 1953. 6. Spector SL, Luparello TJ, Kopetzky MT, Souhrada JF, Kinsman RA: Response of asthmatics to methacholine challenge and suggestion. Am Rev Respir Dis 113:43, 1976. 7. Hume KM. Gandevia B: Forced expiratory volume before and after isoprenaline. Thorax 12:276, 1957. 8. Goldberg I. Cherniack RM: The effect of nebulized bronchodilator delivered with and without IPPB on ventilatory function in chronic obstructive emphysema. Am Rev Respir Dis 91: 13, 1965. 9. Gimeno F, Berg W Chr, Sluiter HJ, Tammeling GJ: Spirometry-induced bronchial obstruction. Am Rev Respir Dis 105:68. 1972. IO. Chai H. Farr RS, Froehlich LA, Mathison DA, McLean JS. Rosenthal RR, Sheffer AL II, Spector SL, Townley RG: Standardization of bronchial inhalation challenge procedures. J ALLERGY CIIN IMMUNOL 56:323, 1975. Il. Pierson DJ, Dick NP, Petty TL: A comparison of spirometric

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values with subjects in standing and sitting positions. Chest 70: 17, 1976. 12. Gervais P, Reinberg A, Gervais C, Smolensky M, DeFrance 0: Twenty-four-hour rhythm in the bronchial hyperreactivity to house dust in asthmatics. J ALLERGV CLIV IMMLINOI. 59:207, 1977. 13. May CD, Williams CS: Further studies concerning the fluctuating insensitivity of peripheral leukocytes to unrelated allergens and the meaning of nonspeciIic “desensitization.” Clin Allergy 3:319, 1973. 14. Claman HN: How corticosteroids work. J AI t ERGY CI IN ~,ViVUNOL 55:145, 1975. 15. Halpern BN, Neveu T, Spector S: On the nature of the chemical mediators involved in anaphylactic reactions in mice. Br J Pharmacol 20:3X9, 1963. 16. Miller MM, Patterson R: Differential airway reactivity to carbachol and physostigmine sulfate in rhesus monkeys with and without reagin-mediated respiratory responses. Int Arch Allergy Appl Immunol 53~349, 1977. 17. Miller MM, Fish JE, Patterson R: Methacholine and physoatigmine airway reactivity in asthmatic and nonasthmatic sub60: 116, 1977. jects. J AL.LERGY CLIN IMMUNOL 18. Perper RJ, Sanda M, Lichtenstein LM: The relationship of in vitro and in viva allergic histamine release: Inhibition in primates by CAMP active agents. Int Arch Allergy 43:837. 1972. 19. Imbeau SA, Harruff R, Hirscher M, Reed CE: Terbutaline’s CI IN IMMUNO~ effects on the allergy skin test. J ALI.ERGY 62: 193. 1978. 20. Chipps BE, Teets KC, Saunders JP, Sobotka AK, Lichtenstein LM: The effects of oral theophylline and terbutaline on skin tests and histamine. J ALLERGV CLIN IMMUNOI. 61: 171. 1978. (Abst.) 21. Spector SL: Effect of beta-adrenergic agents on skin test responses and bronchial challenge responses. Chest 73S:976S. 1978. 22. Rosenthal RR, Norman PS, Summer WR, Permutt S: Role of the parasympathetic system in antigen-induced bronchospasm. J Appl Physiol 42:600, 1977. 23. Muttari A, Ahonen A, Kellomaki L. Kuusisto P, Lehtinen J, Veneskoski T: The effect of ICI 74,917 on asthma and bronchial provocation tests. Clin Allergy 8:281. 1978. 24. Spector SL: Effect of beta-adrenergic agents on skin test responses and bronchial challenge responses. Chest 735~9765. 1978