Hospitalized Cystic Fibrosis Patients with Acute ...

Aerosolized Recombinant Human DNase in

Hospitalized Cystic Fibrosis Patients with Acute Pulmonary Exacerbations R. W. WILMOTT, R. S. AMIN, A. A. COLIN, A. DEVAULT, A. J. DOlOR, H. EIGEN, C. JOHNSON, L. A. LESTER, K. McCOY, L. P. McKEAN, R. MOSS, M. L. NASH, C. PAGEL JUE, W. REGELMANN, D. C. STOKES, and H. J. FUCHS Divisions of Pulmonary Medicine, Children's Hospital Medical Center, Cincinnati, Ohio; Children's Hospital Medical Center, Boston, Massachusetts; New York Medical College, Valhalla, New York; Riley Hospital for Children, Indianapolis, Indiana; Washington University School of Medicine, St. Louis, Missouri; University of Chicago Hospitals and Clinics, Chicago, Illinois; Columbus Children's Hospital, Columbus, Ohio; Emory University, Atlanta, Georgia; Packard Children's Hospital, Palo Alto, California; Medical Affairs, Genentech, Inc., South San Francisco, California; University of Minnesota, Minneapolis, Minnesota; and Vanderbilt University Medical Center, Nashville, Tennessee

The goal of this study was to evaluate the safety and efficacy of recombinant human DNase (rhDNase) in hospitalized patients with cystic fibrosis (CF) experiencing acute pulmonary exacerbations. Eighty patients with documented CF were enrolled at 11 CF centers when admitted for antibiotic therapy. Patients were at least 5 yr old with a forced vital capacity (FVC) ~ 35% of predicted and an oxygen saturation ~ 90% on a fraction of inspired oxygen (Flo2) < 0.5. Patients were randomized to receive rhDNase 2.5 mg in 2.5 ml excipient twice a day (n = 43) or 2.5 ml excipient alone twice daily (n = 37) along with conventional treatment for exacerbations. Administration of rhDNase was not associated with acute adverse events or deaths, and no patients experienced allergic or anaphylactic reactions. Although forced expiratory volume in one second (FEV1) and FVC improved in both treatment groups during the double-blind period, there were no statistically significant differences in the mean change from baseline in FEV1 or FVC between the two groups. rhDNase therapy is safe and well tolerated in CF patients with acute exacerbations requiring hospitalization, but the study did not demonstrate a statistically significant therapeutic effect of rhDNase when added to a regimen of antibiotics and chest physical therapy. Wilmott RW, Amln RS,Colin AA, DeVault A, Dozor AJ, Elgen H, Johnson C, Lester LA, McCoy K, McKean LP, Moss R, Nash ML, Jue CP, Regelmann W, Stokes DC, Fuchs HJ. AerosolIzed recombinant human DNase In hospitalized cystic fibrosis patients with acute pulmonary exacerbations. AM J RESPIR CRIT CARE MED 1996;153:1914-7.

Cystic fibrosis (CF) is a serious, genetic disease caused by a mutation of the CFTR gene located on the long arm of chromosome 7 (1). The disease is inherited as an autosomal recessive disorder that is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and increased concentrations of sodium and chloride in the sweat (2). The most significant clinical complication of CF is obstructive pulmonary disease. Defective adenosine 3':5'-cyclicphosphate (cAMP)-regulated chloride conductance across the apical membrane of respiratory epithelial cells is thought to cause thick, viscid respira-

(Received in original form September 27, 1994 and in revised form July 18, 1995)

This study was made possible through the support of a grant made by Genentech, Inc. All authors and institutional participants, exclusive of the authors from Genentech, Inc., have affirmed that they have no financial interest in Genentech, Inc. Correspondence and requests for reprints should be addressed to Robert W. Wilmott, M.D., Director, Pulmonary Medicine, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039.

Am J Respir Crit Care Med

Vol 153. pp 1914-1917, 1996

tory secretions and impaired mucociliary clearance (3). This primary abnormality of airway function in CF is associated with the development of small airways obstruction early in life and recurrent bacterial infections of the lower airways, particularly with Pseudomonas aeruginosa and Staphylococcus aureus (2). Studies in the 1950s and 1960s demonstrated that large amounts (3 to 14 mg/ml) of DNA are present in purulent CF sputum (4-6). This DNA is primarily derived from the nuclei of degenerating polymorphonuclear neutrophils (7, 8) that are a major part of the inflammatory response in CF, and accounts for much of the viscosity of CF sputum (9, 10). Earlier studies demonstrated that bovine pancreatic deoxyribonuclease I (DNase I) reduced the viscosity of lung secretions (9, 10). However, the bovine preparations of DNase were contaminated with trypsin and chymotrypsin. When used for clinical administration, there were reports of adverse allergic reactions to the aerosolized protein (11, 12), and clinical use of this material (Dornavac or dornase) was discontinued. A promising new approach to the problem of retained airway secretions in CF was made possible by the cloning of human pancreatic DNase I from a human pancreatic complementary DNA (cDNA) library (13). Recombinant human DNase I (rhDNase)

Wilmott, Amin, Colin, et 0/.: Recombinant DNase in Cystic Fibrosis

1915

has been shown to be effective at hydrolyzing DNA and reducing the viscosity of CF sputum in vitro (13). Aerosolized rhDNase therapy was tested in two phase I studies that indicated that it was safe, and that lung function in CF was improved in the short term (14, 15). A phase II study confirmed these findings in a double-blind study of 181 stable outpatients with CF and mild to moderate pulmonary disease (16). During 10d of administration of rhDNase at three doses, 0.6, 2.5, and 10 mg twice daily, patients experienced a statistically significant improvement in forced vital capacity (FVC), forced expiratory volume in one second (FEV.), dyspnea score, and quality of life measures. A 24wk phase III parallel-design, placebo-controlled, double-blind study of rhDNase in 968 adults and children with 'CF was recently completed. The results showed that administration of rhDNase at a dose of 2.5 mg daily or 2.5 mg twice a day significantly reduced the risk of respiratory tract infections and improved the mean FEV. by approximately 6070. The only significant side effect observed was voicealteration, which was increased in rhDNase-treated patients (17). Cystic fibrosis patients suffer from intermittent exacerbations of pulmonary infection that are associated with increased coughing, dyspnea, change in sputum color and composition, and increased sputum bacterial counts. Conventional therapy for such pulmonary exacerbations consists of hospitalization, intensive chest physiotherapy, nutritional support, and intravenous antibiotics. The phase III trial of rhDNase in patients started on rhDNase while stable showed that it could be safely tolerated during pulmonary exacerbations. Therefore, the goal of the present study was to test the hypothesis that initiation of treatment with aerosolized rhDNase in CF patients experiencing an acute pulmonary exacerbation is safe and associated with significantly greater improvements in lung function (FVC, FEV.) and quality of life scores (specifically cough and dyspnea scores).

night stay. A pulmonary exacerbation was defined as the presence of at least four of the following criteria: change in sputum production, new or increased hemoptysis, increased coughing, increased dyspnea, increased fatigue or lethargy, fever greater than 380 C orally, increased chest discomfort, anorexia or weight loss (> 5070 of ideal body weight in 2 wk), reduction by 10070 or more in FEV. (or FVC), new findings on the chest radiograph, or new findings on auscultation of the chest. Patients were treated with either oral ciprofloxacin (to facilitate early discharge), combination intravenous therapy (a J3-lactam and an aminoglycoside) or with intravenous Imipenem-Cilastin sodium. Antibiotics were administered for at least 7 d and changed according to sputum culture and sensitivity results. Approximately 90070 of the patients in each group received an intravenous aminoglycoside, and 65070 in each group received intravenous ceftazidime. Only 8 of 36 placebo and 8 of 44 active patients received oral ciprofloxain. Aminoglycoside dosages were adjusted to achieve therapeutic, nontoxic, peak and trough concentrations. Following admission, patients were maintained on routine chest percussion and postural drainage performed 30 to 60 min before the administration of study drug. They were allowed to maintain routine bronchodilator therapy, which was also given 30 to 60 min before the study drug. Other routine outpatient medications were continued. Randomization was stratified by study center using a permuted block design. The study drug was administered from coded vials containing 2.5 ml excipient with 2.5 mg rhDNase, or 2.5 ml excipient alone. The excipient was composed of sodium chloride (8.77 mg/ml) and calcium chloride (0.15 mg/ml) buffered to a pH of 7.0 ± 1.0. The active treatment was identical in appearance, taste, and odor to the control treatment. Hudson T Up Draft II nebulizers (Hudson RCI, Irvine, CAl and Pulmo-Aide compressors (DeVilbiss, Somerset, PAl were used to administer study drug by aerosol over 10 to 15 min. The dose of rhDNase was chosen based on phase II clinical data (16). Treatments were given twice daily, and the first three doses were given under supervision during the period of hospitalization. The study drug was given each day at the same times (± 2 h) for 14 d. A final evaluation was performed on Day 15. On Days 1,2, 8, and 15, subjects were evaluated by history, physical examination, vital signs, a visual-analog dyspnea score (20), and pulmonary function tests. The visual-analog score allows patients to assess their sense of dyspnea on a loo-mm line between 0 ("no shortness of breath") and 100("short of breath as bad as it can be"). Pulmonary function tests were performed according to standards published by the American Thoracic Society (21). A quality of life questionnaire was administered on Days 1, 8, and 15 (17). There were no modifications to the protocol. An "intent-to-treat" approach was used in the analysis and interpretation of data; all patients randomized to rhDNase or placebo were included in the analysis, regardless of medication compliance or withdrawal from study medication. Data collected were monitored by Genentech Inc., which established a data base and performed the statistical analysis. Results are presented as frequencies and percentages, or means ± standard error values (SE). All statistical tests were two-tailed, and p ~ 0.05 was considered statistically significant. Tests on-means used analysis of variance (ANOVA) or analysis of covariance (ANCOVA) and chi-squared procedures were used on frequencies. '

METHODS A double-blind, parallel, placebo-controlled study was performed at 11 CF centers between September 1992 and December 1992. Eighty patients age 5 yr or more were enrolled at the time of an acute pulmonary exacerbation and randomized to receive either placebo or 2.5 mg rhDNase by aerosol administration twice a day for 14 d. The number of patients was calculated based on the sample size required to have 90070 power to detect a 12070 difference in FEV. improvement between baseline and Day 15 in patients receiving rhDNase. The standard deviation of percent change in FEV. from baseline to Day 10 was 16.0070 in the phase II study (16). Power calculations indicated that 40 patients were required in each group. Inclusion criteria included age greater than 5 yr. The presence of CF was documented by one clinical finding consistent with the diagnosis of CF, together with a sweat sodium or chloride concentration ~ 60 mEq/L by quantitative pilocarpine iontophoresis (18), or homozygosity for the L\F508 gene. Patients were required to have a documented FVC greater than 35070 of the predicted value for gender, age, and height (19) after admission to the hospital and prior to randomization. Oxygen saturations after resting for 10 min were greater or equal to 90070 on a fraction of inspired oxygen (F102) ~ 0.5 (or ~ 85070 at elevations above 4,500 feet). Females of childbearing potential had a negative pregnancy test and were using effective methods of contraception. Patients or their legal guardians provided written informed consent in every case. The research protocol was approved by the institutional review boards of all the participating institutions. Exclusion criteria were the presence of hypercapnia (Paco 2 > 55 mm Hg), respiratory acidosis (pH < 7.3), recent use of narcotics, significant hemoptysis requiring intervention within 180 d of randomization, current pneumothorax requiring a chest tube, a focus of infection outside the respiratory tract, previous treatment with rhDNase, pregnancy, lactation, or a history of malignancy within 5 yr. Patients were enrolled when admitted to the hospital for the treatment of an acute pulmonary exacerbation that required at least one over-

RESULTS All 80 patients completed the study and the baseline characteristics of the two groups were similar (Table 1). The randomization by center resulted in more patients being assigned to the rhDNase group. Table 2 shows the number of patients in each treatment group by center. It was noted that the mean baseline FVC and FEV. values were slightly lower in the rhDNase-treated subjects (p = NS). Analysis of the safety data showed that no patients experienced an acute adverse event, although one rhDNase subject developed a pneumothorax that was considered unrelated to the study drug (p = NS). No patient developed allergicor anaphylactic reactions. An increased proportion of rhDNase-treated patients reported voice alteration (6 of 43 versus 2 of 37; p = NS) compared with placebo-treated patients. There wereno sustained decreases in FEV. in patients treated with rhDNase. Seven pa-

1916

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

TABLE 1

~

BASELINE CHARACTERISTICS OF THE CF PATIENTS WITHIN EACH TREATMENT GROUP*

en

Placebo (n = 37)

2.5 mg rhDNase BID

20.2 ± 1.5 43.2% 56.8% 153.8 ± 2.8

19.6±1.4 55.8% 44.2% 152.6 ± 2.6

(n

= 43)

0 0

5

0

a.

U)

0 c

-5

T

Q)

C>

c

-10

17.5 16.3 39.1 58.0 31.6 94.1

± 0.7 ± 0.6 ± 3.2 ± 3.0 ± 3.2 ± 0.4+

± 0.6 ± 0.5 ± 2.8 ± 2.8 ± 4.4t ± 0.3

..c

0

,

c

«S Q)

.1

""" T

-15

'#. ~

1

*

«S

18.2 17.4 41.1 61.0 36.1 94.4

1996

«S Q)

c

~

Age, yr % Male % Female Height, cm Body mass index, kg/m 2 Males Females FEV1, % predicted Mean FVC, % predicted Mean dyspnea, mm Mean 02 saturation, %

VOL 153

- -- -- --- --- - - --- - - - -- -- -

-.1T

-20 0

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Days on Study

* Values are mean ± SE.

t n = 42. +n = 36.

- -.- -

- - - Placebo

DNase

Figure 2. Mean percent change in visual analog dyspnea score in

tients (two rhDNase-treated and five placebo-treated) experienced a greater than 20070 decrease in FEV. during the admission. At Day 15, FEV. had improved to baseline or greater in the two rhDNase-treated patients and in four of the five placebo-treated patients. There were no consistent changes from baseline in the physical examination or in any of the laboratory studies. Use of rhDNase was not associated with prolongation of hospitalization or of antibiotic usage. There were no study drug discontinuations and no deaths. Analysis of the efficacy data showed that although FEV. and FVC improved in both study groups during the double-blind study, there were no significant differences in the mean percentage improvement in FEV. between the groups (Figure 1). Two placebo-treated patients experienced very large increases in FEV 1 during the study, which created a large standard error of the mean for change in FEV. in the control group. The mean change in dyspnea measured on a visual analog scale improved in both groups of patients (Figure 2) and the percent change was significantly greater in the rhDNase-treated patients at Day 8 (p < 0.01) but not significant at Day 15 (p = 0.10) using an ANCOVA adjusted for baseline differences. The quality of life scores obtained by questionnaire showed no difference 40 ~

> W

LL

30

c Q)

C>

80 cystic fibrosis patients receiving antibiotics for an acute pulmonary exacerbation. Forty-three patients received aerosolized DNase

2.5 mg twice daily (broken line with solid squares), and 37 patients received placebo (solid line with closed circles). Vertical bars represent standard error values. *p < 0.01 by ANCOVA adjusted for baseline values.

between the groups in the amount of improvement in cough and congestion (frequency and severity of coughing spells, lung congestion, and ease of expectoration), dyspnea at different levels of effort, activity limitation in relationship to activities of daily living and intermediate activities, emotional well-being, fatigue, days of restriction to bed, or general health perception. DISCUSSION

Administration of rhDNase to patients with CF during acute pulmonary exacerbations was safe and was not associated with a statistically significant increase in adverse reactions. Six of 43 DNase-treated patients reported voice alteration, and similar symptoms have been reported before in CF patients treated with aerosolized rhDNase (16). It may have resulted from upper airway irritation by rhDNase or from increased mobilization of respiratory secretions and usually resolved in a few days with no change in the administration of study drug. The primary outcome variables, FEV. and FVC, improved in both groups of patients by 10to 15070 with no significant differences between the groups in the mean percent change from base-

20

c

«S

..c

0

TABLE 2

10

NUMBER OF CF PATIENTS IN EACH TREATMENT GROUP BY STUDY SITE

'#. c

«S

0

rhDNase

Q)

~

-10 0

2

3

4

5

6

7

8

9

10 11

12

13

14 15 16

Days on Study - - - Placebo

Figure 1. Mean percent change in FEV, during 14 d of antibiotic therapy in 80 cystic fibrosis patients. Forty-three patients received aerosolized DNase 2.5 mg twice daily (broken line with closed squares), and 37 patients received placebo (solid line with closed circles). Vertical bars represent standard errors.

Children's Hospital Medical Center, Boston Children's Hospital Medical Center, Cincinnati Columbus Children's Hospital Emory University New York Medical College Packard Children's Hospital Riley Hospital for Children University of Chicago University of Minnesota VanderbiltUniversity Washington University Total

5 4

4 5 4 4 2 5

4 4

Placebo

4 2 4 5 4

4 2

4 4 4

2

o

43

37

Wilmott, Amin, Colin, et a/.: Recombinant DNase in Cystic Fibrosis

line. FVC and FEV 1 improvements of this magnitude are typically seen during aggressive treatment of pulmonary exacerbations. Dyspnea was a protocol-defined secondary outcome measure, and there was significantly greater improvement in visual analog dyspnea score in the rhDNase-treated patients at Day 8. At baseline, both groups had analog dyspnea scores of 32 to 36, which represents a moderate clinical degree of dyspnea. The visual analog dyspnea score was validated in earlier studies (16, 20) and has shown a beneficial short-term response to rhDNase (16). The improvement in the actively treated group was by 10070 more than in the control group. This difference is of the order of 0.5 standard deviation, which is a moderate statistical effect size that is likely to be of clinical significance. The dyspnea assessment by quality of life questionnaire improved in both groups of patients, but the change was not statistically significant even though, overall, the two methods for assessment of dyspnea correlated by Spearman's rank order correlation analysis (r = 0.5097, p = 0.0001). It therefore appears that these two methods of assessment are indeed measuring the same subjective complaint but that the visual scale method has greater sensitivity. The results of pulmonary function testing suggest that rhDNase therapy does not add significantly to a standard regimen of antibiotics and chest physical therapy during an acute pulmonary exacerbation of CF. Another explanation for the lack of statistical significance in the analysis of FEVI results by ANOVA could be that the study was under-powered. The assumptions for estimating variance were not met in this study, and two placebo-treated subjects had very large increases in FEV 1. Moreover, the sick CF patients in general had greater variation in pulmonary function values than predicted from the phase II data (16) It is also possible that the dose of rhDNase was too small, although the data available so far on the dose of DNase suggest that 2.5 mg twice daily is an adequate dose in stable patients (16, 17);higher doses may be required to achieve efficacy during acute exacerbations. It appears safe to initiate rhDNase therapy during an acute pulmonary exacerbation, and this would be an appropriate time to start it in a CF patient with chronic airway obstruction for whom rhDNase therapy is planned. However, severely affected individuals have pulmonary function tests that are worse than those in this study, and subjects with FVC < 35070 of the predicted value might respond differently to the treatment. Other multicenter studies of rhDNase in severely ill CF patients are currently in progress, and these will allow a more detailed evaluation of the role of rhDNase therapy in such patients. Acknowledgment: The writers thank the following individuals for their contributions to this study: Stanford University School of Medicine, Stanford, California: Elizabeth A. Harkins, R.N., Ph.D.; Children's Hospital Medical Center, Boston, Massachusetts: Mary-Alice Tully, R.N.C., P.N.P.,and Madeline Knapp, R.N., B.S.; Washington University School of Medicine, St. Louis, Missouri: Mary Boyle, M.S.N.; University of Minnesota, Minneapolis, Minnesota: Jackie Zirbes, R.N., P.N.P., Sue Severson, R.N., Carlos Milia, M.D., Warren Warwick, M.D., and C. Carlyle Clawson, M.D.; New York Medical College, Valhalla, New York: Rose Murray, R.N., B.S., and Gerald Smith, R.R.T.; University of Cincinnati College of Medicine, Cincinnati, Ohio: Vikki Kociela, R.N.; Vanderbilt University Medical Center, Nashville, Tennessee: Judy Marciel, R.N., M.S.N., and Suzanne Campbell, R.R.T.

References 1. Riordan, J. R., J. M. Rornrnens, B. Kerern, N. Alon, R. Rozmahel,

Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J. L. Chou, M.. L.

1917 Drumm, M. C. Iannuzzi, F. S. Collins, and L. C. Tsui. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073. 2. Boat, T. F. 1989. Cystic fibrosis. In C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, editors. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York. 2649-2680. 3. Boucher, R. C., C. U. Cotton, J. T. Gatzy, M. R. Knowles, and J. R. Yankaskas. 1988. Evidence for reduced CI- and increased Na + permeability in cystic fibrosis human primary cell cultures. J. Physiol. 405:77-103. 4. Chernick, W. S., and G. J. Barbero. 1959. Composition of tracheobronchial secretions in cystic fibrosis of the pancreas and bronchiectasis. Pediatrics 24:739-745. 5. Potter, J., L. W. Matthews, J. Lemm, and S. Spector. 1960. The composition of pulmonary secretions from patients with and without cystic fibrosis. Am. J. Dis. Child. 100:493-495. 6. Matthews, L. W., S. Spector, J. Lemm, and J. L. Potter. 1963. Studies on pulmonary secretions: 1. The overall composition of pulmonary secretions from patients with cystic fibrosis, bronchiectasis and laryngectomy. Am. Rev. Respir. Dis. 88:199-204. 7. Potter, J. L., S. Spector, L. W. Matthews, and J. Lemm. 1969. Studies on pulmonary secretions. 3. The nucleic acids in whole pulmonary secretions from patients with cystic fibrosis, bronchiectasis, and laryngectomy. Am. Rev. Respir. Dis. 99:909-916. 8. Lethem, M. I., S. L. James, C. Marriott, and J. F. Burke. 1990. The origin of DNA associated with mucus glycoproteins in cystic fibrosis sputum. Eur. Respir. J. 3:19-23. 9. Armstrong, J. B., and J. C. White. 1950.Liquefaction of viscouspurulent exudates by deoxyribonuclease. Lancet 2:739-742. 10. Chernick, W. S., G. J. Barbero, and H. J. Eichel. 1961. In vitro evaluation of effect of enzymes on tracheobronchial secretions from patients with cystic fibrosis. Pediatrics 27:589-596. 11. Spier, R., E. Witebsky, and J. R. Paine. 1961. Aerosolized pancreatic dornase and antibiotics in pulmonary infections. J.A.M.A. 178: 110-118. 12. Raskin, P. 1968. Bronchospasm after inhalation of pancreatic dornase. Am. Rev. Respir. Dis. 98:597-598. 13. Shak, S., D. J. Capon, R. Hellmiss, S. A. Marsters, and C. L. Baker. 1990. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc. Natl. A cad. Sci. U.S.A. 87:9188-9192. 14. Aitken, M. L., W. Burke, G. McDonald, S. Shak, A. B. Montgomery, and A. Smith. 1992. Recombinant human DNase inhalation in normal subjects and patients with cystic fibrosis. A phase 1 study. J.A.M.A. 267:1947-1951. 15. Hubbard, R. C., N. G. McElvaney, P. Birrer, S. Shak, W. W. Robinson, C. Jolley, M. Wu, M. S. Chernick, and R. G. Crystal. 1992. A preliminary study of aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis. N. Engl. J. Med. 326: 812-815. 16. Ramsey, B. W., S. J. Astley, M. L. Aitken, W. Burke, A. A. Colin, H. L. Dorkin, J. D. Eisenberg, R. L. Gibson, I. R. Harwood, D. V. Schidlow, R. W. Wilmott, M. E. Wohl, L. J. Meyerson, S. Shak, H. Fuchs, and A. L. Smith. 1993. Efficacy and safety of short-term administration of aerosolized recombinant human deoxyribonuclease in patients with cystic fibrosis. Am. Rev. Respir. Dis. 148:145-151. 17. Fuchs, H. J., D. S. Borowitz, D. H. Christiansen, E. M. Morris, M. L. Nash, B. W. Ramsey, B. J. Rosenstein, A. L. Smith, and M. E. Wohl. 1994. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N. Engl. J. Med 331:637-642. 18. Gibson, L. E., and R. E. Cooke. 1959. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23:545-549. 19. Knudson, R. J., M. D. Lebowitz, C. J. Holberg, and B. Burrows. 1983. Changes in the normal maximal expiratory flow-volume curve with growth and aging. Am. Rev. Respir. Dis. 127:725-734. 20. Gift, A. G. 1989. Validation of a vertical visual analogue scale as a measure of clinical dyspnea. Rehabil. Nurs. 14:323-325. 21. American Thoracic Society. 1987.Standardization of spirometry - 1987 update. Statement of the American Thoracic Society. Am. Rev. Respir. Dis. 136:1285-1298.