Hypertonic Saline for Cystic Fibrosis
http://www.100md.com
《新英格兰医药杂志》
To the Editor: We question the selection by Elkins et al.1 and Donaldson et al.2 (Jan. 19 issue) of 7 percent hypertonic saline, which can result in bronchoconstriction, in these studies of therapy for cystic fibrosis. Elkins et al. report a fall of 94 ml in the forced expiratory volume in one second (FEV1) after the first dose of medication, which is greater than the reported final improvement in FEV1 of 68 ml. Conversely, Donaldson et al. do not specify any change in FEV1 with the use of 7 percent hypertonic saline. Robinson et al.3 have compared mucociliary clearance with the use of different concentrations of hypertonic saline and did not find any difference in efficacy between solutions of 3 percent and 7 percent hypertonic saline solutions. We have shown that the use of 3 percent hypertonic saline is effective and has the additional advantage of not causing a substantial change in FEV1, oxygen saturation, or symptom score.4 Hence, the choice of the strength of the hypertonic saline solution administered should be based on the potential effects of hypertonic saline on pulmonary function, oxygen saturation, palatability, and the patient's preference.
Imran Aziz, M.B., B.S.
Royal Albert Edward Infirmary
Wigan WN1 2NN, United Kingdom
Jack A. Kastelik, M.D.
Castle Hill Hospital
Cottingham HU16 5JQ, United Kingdom
j.a.kastelik@hull.ac.uk
References
Elkins MR, Robinson M, Rose BR, et al. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006;354:229-240.
Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006;354:241-250.
Robinson M, Hemming AL, Regnis JA, et al. Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis. Thorax 1997;52:900-903.
Kastelik JA, Aziz I, Morice AH. Sputum induction in young cystic fibrosis patients. Eur Respir J 2001;17:832-832.
To the Editor: Donaldson and colleagues report that hypertonic saline after pretreatment with amiloride did not result in a sustained increase in mucus clearance or improvement in lung function or respiratory symptoms because of inhibition of apical membrane water permeability. Animal airways have a moderate osmotic water permeability and express aquaporin water channels, one of which is aquaporin-3.1,2,3 In Table 1 of their article, the authors report that 50 percent of the patients in each of the two study groups received inhaled steroids concomitantly. Corticosteroids have been found to induce the expression of aquaporin-3 in A549 cells, a human airway epithelial-cell line derived from lung adenocarcinoma, in vitro.3 In addition, hypertonicity induces the expression of aquaporin-3 in Madin–Darby canine-kidney cells, a renal epithelial-cell line, in vitro.4 Perhaps patients receiving concomitant treatment with inhaled steroids should have been studied separately, in order to identify the possible contribution of aquaporin-3 overexpression to hypertonic saline treatment.
Sotirios Zarogiannis, B.Sc.
Chrissi Hatzoglou, M.D., Ph.D.
Konstantinos Gourgoulianis, M.D., Ph.D.
University of Thessaly
41222 Larissa, Greece
szarog@med.uth.gr
References
Verkman AS. Role of aquaporin water channels in kidney and lung. Am J Med Sci 1998;316:310-320.
Verkman AS, Matthay MA, Song Y. Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 2000;278:L867-L879.
Tanaka M, Inase N, Fushimi K, et al. Induction of aquaporin 3 by corticosteroid in a human airway epithelial cell line. Am J Physiol 1997;273:L1090-L1095.
Matsuzaki T, Suzuki T, Takata K. Hypertonicity-induced expression of aquaporin 3 in MDCK cells. Am J Physiol Cell Physiol 2001;281:C55-C63.
To the Editor: The importance of the volume of the airway surface liquid in the pathophysiology of cystic fibrosis lung disease is supported by the findings of Elkins et al. and Donaldson et al. As pointed out in the accompanying editorial by Ratjen,1 the mechanism of the prolonged action of inhaled hypertonic saline remains to be elucidated. We suggest that one mechanism pertains not to the volume of the airway surface liquid but, rather, to the effect of sodium ions on the viscosity of the mucus gel. Studies of gastrointestinal mucins have shown that calcium is the main cation that binds to mucins; the interaction increases the viscosity of the mucus gel.2 Calcium binding to mucin is displaced by hypertonic sodium chloride.3 Whether these observations pertain to airway mucins in patients with cystic fibrosis requires further investigation. Since mucus hyperviscosity has been implicated in the intestinal, hepatobiliary, and pancreatic manifestations of cystic fibrosis, hypertonic saline might be useful for the prevention of complications in these organs as well.
Rahul Kuver, M.D.
Sum P. Lee, M.D., Ph.D.
University of Washington
Seattle, WA 98195
kuver@u.washington.edu
References
Ratjen F. Restoring airway surface liquid in cystic fibrosis. N Engl J Med 2006;354:291-293.
Forstner JF, Forstner GG. Calcium binding to intestinal goblet cell mucin. Biochim Biophys Acta 1975;386:283-292.
Kuver R, Lee SP. Calcium binding to biliary mucins is dependent on sodium ion concentration: relevance to cystic fibrosis. Biochem Biophys Res Commun 2004;314:330-334.
Drs. Bye and Elkins reply: Drs. Aziz and Kastelik question the selection of a concentration higher than 3 percent in our phase 3 trial of inhaled hypertonic saline for cystic fibrosis. On the basis of the single-intervention studies they cite, we agree that a single dose of 3 percent saline increases the weight of sputum expectorated and improves mucociliary clearance to a degree similar to 7 percent saline. However, the primary outcome of our trial was lung function, which was chosen because it correlates with mortality in patients with cystic fibrosis.1 When designing the trial, we therefore also considered the data from phase 2 trials that examined the effect of the regular use of hypertonic saline on lung function. We were unable to find evidence of an improvement in lung function with 3 percent saline. At higher concentrations, however, there was evidence of a benefit in both mucociliary clearance and lung function.2 Our observations that 7 percent saline did not result in excessive side effects in clinical practice and in previous trials were supported by others.3 We therefore chose 7 percent saline as the intervention for our trial. Further studies comparing various concentrations, as well as dosages and delivery systems, would help to refine the treatment protocol.
Drs. Aziz and Kastelik also express concern that the 94-ml fall in FEV1 after the first dose of hypertonic saline was greater than the final improvement in FEV1, of 68 ml. As stated in the article, premedication with a bronchodilator resulted in a 60-ml improvement in FEV1 that limited the effective fall from baseline. We also stated that the final improvement of 68 ml was relative to baseline. Thus, any initial fall in FEV1 was recovered, and then an additional average improvement of 68 ml was achieved.
Drs. Kuver and Lee suggest that a possible mechanism of action of hypertonic saline in the lungs is the effect of sodium ions on the viscosity of the airway mucus gel. Other authors have examined this possibility, as mentioned in our article and as reviewed more comprehensively by King.4 Our trial did not provide any data to indicate whether hypertonic saline would have an effect on mucins from other organs.
Peter T.P. Bye, Ph.D.
Mark R. Elkins, M.H.Sc.
Royal Prince Alfred Hospital
Sydney, NSW 2050, Australia
peterb@med.usyd.edu.au
References
Milla CE, Warwick WJ. Risk of death in cystic fibrosis patients with severely compromised lung function. Chest 1998;113:1230-1234.
Eng PA, Morton J, Douglass JA, Riedler J, Wilson J, Robertson CF. Short-term efficacy of ultrasonically nebulized hypertonic saline in cystic fibrosis. Pediatr Pulmonol 1996;21:77-83.
Suri R, Metcalfe C, Lees B, et al. Comparison of hypertonic saline and alternate-day or daily recombinant human deoxyribonuclease in children with cystic fibrosis: a randomised trial. Lancet 2001;358:1316-1321.
King M. Mucolytics and mucus clearance. In: Rubin BK, van der Schans CP, eds. Therapy for mucus-clearance disorders. Vol. 188 of Lung biology in health and disease. New York: Marcel Dekker, 2004:201-24.
Dr. Donaldson and colleagues reply: Mr. Zarogiannis et al. point out that corticosteroids and hypertonicity up-regulate aquaporin-3 and speculate that the use of inhaled corticosteroids could have influenced responses to amiloride, hypertonic saline, or both. Because the effect of amiloride on water transport was discovered only after the completion of our clinical trial, we neither excluded nor studied separately patients receiving inhaled corticosteroids. Reassuringly, inhaled corticosteroid use was balanced in the randomized groups, and all subjects were exposed to hypertonic saline, making it unlikely that an effect on aquaporin-3 greatly influenced the trial outcomes. Finally, recent in vitro experiments in our laboratory (unpublished data) suggest that water transport by means of aquaporin-3 is not attenuated by amiloride.
Drs. Aziz and Kastelik question the selection of 7 percent (as compared with 3 percent) saline. They refer to their own study, which reported safety with a single dose of 3 percent saline used for sputum induction. Because the mass of salt deposited on airway surfaces determines the magnitude of the increase in the volume of airway surface liquid, we sought to use the highest concentration of hypertonic saline that would be safe and well tolerated. Robinson et al.1 provided good evidence for a dose–effect relationship between hypertonic saline and mucociliary clearance, despite the absence of a significant difference between the 3 percent and 7 percent groups. Twelve percent saline was poorly tolerated, however, because of oropharyngeal irritation. Therefore, 7 percent saline was selected on the basis of the study by Robinson et al. and other short-term studies of hypertonic saline in cystic fibrosis. In our study, we report FEV1 values at two hours after administration of the first dose of hypertonic saline — values that, in fact, increased from baseline, further supporting the assertion that 7 percent saline is well tolerated in patients with cystic fibrosis.
Drs. Kuver and Lee propose an alternative mechanism linking the use of hypertonic saline and stimulated mucociliary clearance. Displacement by sodium of the calcium ions that bind mucins is postulated to explain the expulsion of mucins during exocytosis, and may influence the rheologic properties of mucus once secreted.2 In fact, we did invoke the "electrostatic effects" of hypertonic saline to explain acutely stimulated mucociliary clearance after amiloride plus inhalation of hypertonic saline, because in vitro data suggested that little increase in airway surface liquid volume occurs in this situation because amiloride blocks water transport. During treatment with hypertonic saline without amiloride, however, isotonicity is restored rapidly (in approximately two minutes) in the airway lumen,3 suggesting that both the acute and the sustained effects on mucociliary clearance of placebo or hypertonic saline were due to improved hydration of secretions, rather than to the persistence of a high salt environment.
Scott H. Donaldson, M.D.
Robert Tarran, Ph.D.
Richard C. Boucher, M.D.
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
scott_donaldson@med.unc.edu
References
Robinson M, Hemming AL, Regnis JA, et al. Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis. Thorax 1997;52:900-903.
Verdugo P. Mucin exocytosis. Am Rev Respir Dis 1991;144:S33-S37.
Tarran R, Grubb BR, Parsons D, et al. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell 2001;8:149-158.
Dr. Ratjen replies: Drs. Kuver and Lee propose that the beneficial effect of inhaled hypertonic saline in cystic fibrosis may be due to the displacement by sodium of calcium ions bound to mucins, thereby reducing the viscosity of airway mucus. Hypertonic saline has indeed been shown to improve sputum rheology, and it is conceivable that changes in mucin ion composition contribute to this finding.1 However, changes in the mechanical properties of mucus would not explain the prolonged effect of hypertonic saline on airway surface liquid height in vitro, since these experiments were performed in the absence of a mucus layer. In addition, agents that merely change the rheology of airway secretions do not affect mucociliary clearance in cystic fibrosis. This is highlighted by studies with recombinant human DNase, which reduces sputum viscosity but, unlike hypertonic saline, does not increase mucociliary clearance.2,3 These observations would therefore support the concept that hypertonic saline, rather than acting primarily as a mucolytic agent, improves mucociliary clearance through an increase in airway surface liquid height.
Felix Ratjen, M.D., Ph.D.
Hospital for Sick Children
Toronto, ON M5G 1X8, Canada
References
King M, Dasgupta B, Tomkiewicz RP, Brown NE. Rheology of cystic fibrosis sputum after in vitro treatment with hypertonic saline alone and in combination with recombinant human deoxyribonuclease I. Am J Respir Crit Care Med 1997;156:173-177.
Laube BL, Auci RM, Shields DE, et al. Effect of rhDNase on airflow obstruction and mucociliary clearance in cystic fibrosis. Am J Respir Crit Care Med 1996;153:752-760.
Robinson M, Hemming AL, Moriarty C, Eberl S, Bye PT. Effect of a short course of rhDNase on cough and mucociliary clearance in patients with cystic fibrosis. Pediatr Pulmonol 2000;30:16-24.
Imran Aziz, M.B., B.S.
Royal Albert Edward Infirmary
Wigan WN1 2NN, United Kingdom
Jack A. Kastelik, M.D.
Castle Hill Hospital
Cottingham HU16 5JQ, United Kingdom
j.a.kastelik@hull.ac.uk
References
Elkins MR, Robinson M, Rose BR, et al. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006;354:229-240.
Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006;354:241-250.
Robinson M, Hemming AL, Regnis JA, et al. Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis. Thorax 1997;52:900-903.
Kastelik JA, Aziz I, Morice AH. Sputum induction in young cystic fibrosis patients. Eur Respir J 2001;17:832-832.
To the Editor: Donaldson and colleagues report that hypertonic saline after pretreatment with amiloride did not result in a sustained increase in mucus clearance or improvement in lung function or respiratory symptoms because of inhibition of apical membrane water permeability. Animal airways have a moderate osmotic water permeability and express aquaporin water channels, one of which is aquaporin-3.1,2,3 In Table 1 of their article, the authors report that 50 percent of the patients in each of the two study groups received inhaled steroids concomitantly. Corticosteroids have been found to induce the expression of aquaporin-3 in A549 cells, a human airway epithelial-cell line derived from lung adenocarcinoma, in vitro.3 In addition, hypertonicity induces the expression of aquaporin-3 in Madin–Darby canine-kidney cells, a renal epithelial-cell line, in vitro.4 Perhaps patients receiving concomitant treatment with inhaled steroids should have been studied separately, in order to identify the possible contribution of aquaporin-3 overexpression to hypertonic saline treatment.
Sotirios Zarogiannis, B.Sc.
Chrissi Hatzoglou, M.D., Ph.D.
Konstantinos Gourgoulianis, M.D., Ph.D.
University of Thessaly
41222 Larissa, Greece
szarog@med.uth.gr
References
Verkman AS. Role of aquaporin water channels in kidney and lung. Am J Med Sci 1998;316:310-320.
Verkman AS, Matthay MA, Song Y. Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 2000;278:L867-L879.
Tanaka M, Inase N, Fushimi K, et al. Induction of aquaporin 3 by corticosteroid in a human airway epithelial cell line. Am J Physiol 1997;273:L1090-L1095.
Matsuzaki T, Suzuki T, Takata K. Hypertonicity-induced expression of aquaporin 3 in MDCK cells. Am J Physiol Cell Physiol 2001;281:C55-C63.
To the Editor: The importance of the volume of the airway surface liquid in the pathophysiology of cystic fibrosis lung disease is supported by the findings of Elkins et al. and Donaldson et al. As pointed out in the accompanying editorial by Ratjen,1 the mechanism of the prolonged action of inhaled hypertonic saline remains to be elucidated. We suggest that one mechanism pertains not to the volume of the airway surface liquid but, rather, to the effect of sodium ions on the viscosity of the mucus gel. Studies of gastrointestinal mucins have shown that calcium is the main cation that binds to mucins; the interaction increases the viscosity of the mucus gel.2 Calcium binding to mucin is displaced by hypertonic sodium chloride.3 Whether these observations pertain to airway mucins in patients with cystic fibrosis requires further investigation. Since mucus hyperviscosity has been implicated in the intestinal, hepatobiliary, and pancreatic manifestations of cystic fibrosis, hypertonic saline might be useful for the prevention of complications in these organs as well.
Rahul Kuver, M.D.
Sum P. Lee, M.D., Ph.D.
University of Washington
Seattle, WA 98195
kuver@u.washington.edu
References
Ratjen F. Restoring airway surface liquid in cystic fibrosis. N Engl J Med 2006;354:291-293.
Forstner JF, Forstner GG. Calcium binding to intestinal goblet cell mucin. Biochim Biophys Acta 1975;386:283-292.
Kuver R, Lee SP. Calcium binding to biliary mucins is dependent on sodium ion concentration: relevance to cystic fibrosis. Biochem Biophys Res Commun 2004;314:330-334.
Drs. Bye and Elkins reply: Drs. Aziz and Kastelik question the selection of a concentration higher than 3 percent in our phase 3 trial of inhaled hypertonic saline for cystic fibrosis. On the basis of the single-intervention studies they cite, we agree that a single dose of 3 percent saline increases the weight of sputum expectorated and improves mucociliary clearance to a degree similar to 7 percent saline. However, the primary outcome of our trial was lung function, which was chosen because it correlates with mortality in patients with cystic fibrosis.1 When designing the trial, we therefore also considered the data from phase 2 trials that examined the effect of the regular use of hypertonic saline on lung function. We were unable to find evidence of an improvement in lung function with 3 percent saline. At higher concentrations, however, there was evidence of a benefit in both mucociliary clearance and lung function.2 Our observations that 7 percent saline did not result in excessive side effects in clinical practice and in previous trials were supported by others.3 We therefore chose 7 percent saline as the intervention for our trial. Further studies comparing various concentrations, as well as dosages and delivery systems, would help to refine the treatment protocol.
Drs. Aziz and Kastelik also express concern that the 94-ml fall in FEV1 after the first dose of hypertonic saline was greater than the final improvement in FEV1, of 68 ml. As stated in the article, premedication with a bronchodilator resulted in a 60-ml improvement in FEV1 that limited the effective fall from baseline. We also stated that the final improvement of 68 ml was relative to baseline. Thus, any initial fall in FEV1 was recovered, and then an additional average improvement of 68 ml was achieved.
Drs. Kuver and Lee suggest that a possible mechanism of action of hypertonic saline in the lungs is the effect of sodium ions on the viscosity of the airway mucus gel. Other authors have examined this possibility, as mentioned in our article and as reviewed more comprehensively by King.4 Our trial did not provide any data to indicate whether hypertonic saline would have an effect on mucins from other organs.
Peter T.P. Bye, Ph.D.
Mark R. Elkins, M.H.Sc.
Royal Prince Alfred Hospital
Sydney, NSW 2050, Australia
peterb@med.usyd.edu.au
References
Milla CE, Warwick WJ. Risk of death in cystic fibrosis patients with severely compromised lung function. Chest 1998;113:1230-1234.
Eng PA, Morton J, Douglass JA, Riedler J, Wilson J, Robertson CF. Short-term efficacy of ultrasonically nebulized hypertonic saline in cystic fibrosis. Pediatr Pulmonol 1996;21:77-83.
Suri R, Metcalfe C, Lees B, et al. Comparison of hypertonic saline and alternate-day or daily recombinant human deoxyribonuclease in children with cystic fibrosis: a randomised trial. Lancet 2001;358:1316-1321.
King M. Mucolytics and mucus clearance. In: Rubin BK, van der Schans CP, eds. Therapy for mucus-clearance disorders. Vol. 188 of Lung biology in health and disease. New York: Marcel Dekker, 2004:201-24.
Dr. Donaldson and colleagues reply: Mr. Zarogiannis et al. point out that corticosteroids and hypertonicity up-regulate aquaporin-3 and speculate that the use of inhaled corticosteroids could have influenced responses to amiloride, hypertonic saline, or both. Because the effect of amiloride on water transport was discovered only after the completion of our clinical trial, we neither excluded nor studied separately patients receiving inhaled corticosteroids. Reassuringly, inhaled corticosteroid use was balanced in the randomized groups, and all subjects were exposed to hypertonic saline, making it unlikely that an effect on aquaporin-3 greatly influenced the trial outcomes. Finally, recent in vitro experiments in our laboratory (unpublished data) suggest that water transport by means of aquaporin-3 is not attenuated by amiloride.
Drs. Aziz and Kastelik question the selection of 7 percent (as compared with 3 percent) saline. They refer to their own study, which reported safety with a single dose of 3 percent saline used for sputum induction. Because the mass of salt deposited on airway surfaces determines the magnitude of the increase in the volume of airway surface liquid, we sought to use the highest concentration of hypertonic saline that would be safe and well tolerated. Robinson et al.1 provided good evidence for a dose–effect relationship between hypertonic saline and mucociliary clearance, despite the absence of a significant difference between the 3 percent and 7 percent groups. Twelve percent saline was poorly tolerated, however, because of oropharyngeal irritation. Therefore, 7 percent saline was selected on the basis of the study by Robinson et al. and other short-term studies of hypertonic saline in cystic fibrosis. In our study, we report FEV1 values at two hours after administration of the first dose of hypertonic saline — values that, in fact, increased from baseline, further supporting the assertion that 7 percent saline is well tolerated in patients with cystic fibrosis.
Drs. Kuver and Lee propose an alternative mechanism linking the use of hypertonic saline and stimulated mucociliary clearance. Displacement by sodium of the calcium ions that bind mucins is postulated to explain the expulsion of mucins during exocytosis, and may influence the rheologic properties of mucus once secreted.2 In fact, we did invoke the "electrostatic effects" of hypertonic saline to explain acutely stimulated mucociliary clearance after amiloride plus inhalation of hypertonic saline, because in vitro data suggested that little increase in airway surface liquid volume occurs in this situation because amiloride blocks water transport. During treatment with hypertonic saline without amiloride, however, isotonicity is restored rapidly (in approximately two minutes) in the airway lumen,3 suggesting that both the acute and the sustained effects on mucociliary clearance of placebo or hypertonic saline were due to improved hydration of secretions, rather than to the persistence of a high salt environment.
Scott H. Donaldson, M.D.
Robert Tarran, Ph.D.
Richard C. Boucher, M.D.
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
scott_donaldson@med.unc.edu
References
Robinson M, Hemming AL, Regnis JA, et al. Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis. Thorax 1997;52:900-903.
Verdugo P. Mucin exocytosis. Am Rev Respir Dis 1991;144:S33-S37.
Tarran R, Grubb BR, Parsons D, et al. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell 2001;8:149-158.
Dr. Ratjen replies: Drs. Kuver and Lee propose that the beneficial effect of inhaled hypertonic saline in cystic fibrosis may be due to the displacement by sodium of calcium ions bound to mucins, thereby reducing the viscosity of airway mucus. Hypertonic saline has indeed been shown to improve sputum rheology, and it is conceivable that changes in mucin ion composition contribute to this finding.1 However, changes in the mechanical properties of mucus would not explain the prolonged effect of hypertonic saline on airway surface liquid height in vitro, since these experiments were performed in the absence of a mucus layer. In addition, agents that merely change the rheology of airway secretions do not affect mucociliary clearance in cystic fibrosis. This is highlighted by studies with recombinant human DNase, which reduces sputum viscosity but, unlike hypertonic saline, does not increase mucociliary clearance.2,3 These observations would therefore support the concept that hypertonic saline, rather than acting primarily as a mucolytic agent, improves mucociliary clearance through an increase in airway surface liquid height.
Felix Ratjen, M.D., Ph.D.
Hospital for Sick Children
Toronto, ON M5G 1X8, Canada
References
King M, Dasgupta B, Tomkiewicz RP, Brown NE. Rheology of cystic fibrosis sputum after in vitro treatment with hypertonic saline alone and in combination with recombinant human deoxyribonuclease I. Am J Respir Crit Care Med 1997;156:173-177.
Laube BL, Auci RM, Shields DE, et al. Effect of rhDNase on airflow obstruction and mucociliary clearance in cystic fibrosis. Am J Respir Crit Care Med 1996;153:752-760.
Robinson M, Hemming AL, Moriarty C, Eberl S, Bye PT. Effect of a short course of rhDNase on cough and mucociliary clearance in patients with cystic fibrosis. Pediatr Pulmonol 2000;30:16-24.