MUCOLYTICS, PULMOZYME

1961 Chernick WS, Barbero GJ, Eichel HJ. In vitro evaluation of the effect of enzymes on tracheobronchial secretions from patients with cystic fibrosis. Pediatrics 1961; 27:589-596. [PubMed]
Effect of various enzymes on the viscosity of CF sputum showed that pancreatic dornase had the most marked effect showing complete dissolution of the fibrous structure of the sputum. This finding supported the theory that the excessive viscosity of CF sputum (figure 1) was related to a fibrous network observed in the sputum which was primarily of deoxyribonucleoproteins as had been shown by this group (Chernick et al, 1959 above).

Figure 1: Viscid sputum from a person with cystic fibrosis.

The effect of enzymes on the sputum had been reported in other chronic pulmonary conditions (Sherry S et al. Proc Soc Exp Biol Med 1948; 68:179) and also on CF sputum (Shwachman & Leubner, 1955 above). These and other early studies eventually led to the introduction of rhDNase (Pulmozyme) as an effective mucolytic but only after the side effects of the biological product were circumvented by producing this genetically engineered product rhDNase (Pulmozyme) (Shak et al, 1990 below). Armstrong JB and White JC (Lancet 1950; 2:739 above) had shown that deoxyribonuclease would liquefy viscous purulent exudate; later Elmes PC & Armstrong JB (Thorax 1953; 8:295-300) reported its use in chronic bronchitis, but the side effects of the bovine preparation precluded its use and further development (Raskin P. Am Rev Respir Dis 1968; 98:697-698). Shwachman (1955 above) mentions that May and Lowe used a pancreatic preparation by inhalation in one patient and that at autopsy widespread granulomatous lesions were found in the lung, a lesion not seen in any other patient. However, eventually Shak et al (1990 below) cloned, sequenced, and expressed rhDNase which later became one of the major advances in treatment of the Nineties (Fuchs et al. 1994 below).

1961 Reid L. The composition of tracheobronchial secretions in cystic fibrosis. Postgrad Med J 1961; 37:599-600. [PubMed]
Her first academic appointment was as a research assistant at the Institute of Diseases of the Chest at London University. She was appointed a professor of experimental pathology at London University in 1967, and was made Dean of the Cardiothoracic Institute at London University in 1973. She accepted a position on the faculty of Harvard Medical School in 1976, as one of the school's few women faculty members. Most of her publications concerned the bronchial secretions and the characteristics of bronchial mucous glands. These were popular areas for research for the few researchers involved in CF at the time, so copious and so very viscid was the sputum of people with cystic fibrosis. As it eventually turned out there were no intrinsic structural abnormalities of the mucus. During her time in the UK she was a good friend to and closely involved with the UK Cystic Fibrosis Research Trust. Lynne Reid reviewed much of her research in 1981 at the Minnesota Meeting (1000 Years of Cystic Fibrosis. University of Minnesota, 1981).

Figure 2: Professor Lynne Reid. From www.nlm.nih.gov/changingthefaceofmedicine.

1962 Lieberman J. Enzymatic dissolution of pulmonary secretions. An in vitro study of sputum from patients with cystic fibrosis of the pancreas. Am J Dis Child 1962; 104: 342-348.
Dr Jack Lieberman of Los Angeles observed that a favourable effect on sputum viscosity by the addition of enzymes had been reported both in vitro (Chernick et al, Pediatrics 1961; 27:589 above) and in vivo (Shwachman et al. Pediatrics 1960; 25:155). This present study confirmed the liquefaction which occurred in the presence of various enzymes in order of effect – trypsin, ficin, chymotrypsin, deoxyribonuclease and elastase.
There was an interesting editorial comment to this article as follows – “Now if someone can find a way to bring a sufficient quantity of a non-irritating mixture of enzymes to bear on the bronchiolar mucus he can see if it works in the patient”. This did eventually occur with rhDNase (Pulmozyme) but it was some 30 years before this was achieved (Shack et al. 1990; Fuchs et al, 1994 below). (Also Lieberman J. JAMA 1968; 205:312-313 below).

1962 Webb WR. Clinical evaluation of a new mucolytic agent acetyl-cysteine. J Thorac Cardiovasc Surg 1962; 44:330-343. [PubMed]
Acetyl cysteine, a derivative of the amino acid cysteine, is a drug that subsequently was widely used for CF in Europe but never popular in the UK. Here experience is reported of 285 patients with a variety of suppurative pulmonary conditions and “revealed it to be extremely effective with almost no associated complications. Seven patients with CF improved over 10 months with either inhalations or sleeping in a tent with the drug nebulised with saline and propylene glycol”. (figure 9).
Apparently N-acetylcysteine and other sulphydryl compounds act by depolymerising mucus in vitro by breaking disulphide bonds of the glycoproteins thereby lowering the viscosity (Sheffner AL. Ann N Y Acad Sci 1963; 106:298-310).

Figure 9: 24 hour sputum collections from a man with purulent bronchitis during the day prior to and the 4 days subsequent to use of acetyl-cysteine.

This paper contains a useful summary of all the many side effects of animal-derived trypsin and enzymatic treatments used to liquefy sputum – contrasted with the minimal side effects experienced with N-acetylcysteine. Interestingly there has been a renewal of interest in acetyl cysteine in the Millennium and the drug has been used both for respiratory (6.5% of USA patients with CF take inhaled acetyl cysteine) and gastrointestinal problems in CF (Lillibridge CB et al. Oral administration of n-acetyl cysteine in the prophylaxis of "meconium ileus equivalent" J Pediatr 1967; 71:887-9; Gracey M et al. Treatment of abdominal pain in cystic fibrosis by oral administration of n-acetyl cysteine. Arch Dis Child 1969; 44:404-405).
More recently the relation to glutathione, in particular, has featured in recent references as N-acetylcysteine is an effective precursor of cysteine for tissue glutathione synthesis. Apparently CFTR is responsible for glutathione transport and there may be intracellular accumulation of glutathione in cystic fibrosis (Childers M. Medical Hypotheses 2007; 68:101-102.)

1963 Denton R. The rheology of human mucus. Ann N Y Acad Sci 1963; 106:746-754. [PubMed]
This is a relevant paper as the work of Robert Denton was important in the introduction of the mist tent (see Denton 1955 above). The paper concludes with the seemingly obvious statement that the degree of flow resistance will show as quantitavely the degree of abnormality and may provide information which can lead to further understanding of the chemical changes within the mucus blanket which are responsible for impaired bronchial drainage.

1963 Reas HW. The effect of N-acetylcysteine on the viscosity of tracheobronchial secretions in cystic fibrosis of the pancreas. J Pediatr 1963; 62:31-5. [PubMed] (Also Reas HW. South Med J. 1963; 56:1271-1278.) [PubMed]
Viscosity measurements were determined on secretions obtained via tracheotomies from 2 patients with CF with the use of N-acetylcysteine aerosol which produced a greater fall in the viscosity of the secretions than did a control aerosol (also Webb 1962 above). The authors suggest that “The combination of this safe method of mucolysis with energetic postural drainage and physiotherapy may be very rewarding in the easier removal of retained pulmonary secretions”. (Also Suddarth SB. Acetylcysteine a new and effective mucolytic agent. Bulletin – Grainger Medical Center 1963; 15:65-69 above; Meeker IA Jr, Kincannon WN. Acetyl cysteine used to liquefy inspissated meconium causing intestinal obstruction in the newborn. Surgery, St Louis 1964; 56:419-425).

Obviously at the time there was considerable interest in this new mucolytic agent whose free sulphydryl group reduced the disulphide linkages of mucoproteins. Subsequently it was the subject of a number of publications in liquefying sputum, improving abdominal pain and treating meconium ileus but never became popular in the UK as part of the pulmonary treatment. Although a subsequent review found no evidence of benefit (Duijvestijn YC & Brand PL. Acta Paediatr 1999; 88:38-41), Ratjen et al (Eur J Pediatr 1985; 144:374-378) found in a 12 week oral trial of NAC, ambroxal and placebo that “although no clinical differences could be observed between the three groups, significant impairment in the placebo group was found for trapped air and FEV1 when compared to the active groups, suggesting a therapeutic effect of ambroxal and NAC in CF”. More recently the relation to glutathione has caused renewed interest in the drug which, when given orally in high doses, provides a source of glutathione and apparently reduces airway inflammation (Tirouvanziam R, et al. Proc Nat Acad Sc 2006; 103:4628-4633).

1963 Chernick WS, Barbero GJ. Studies on human tracheobronchial and submaxillary secretions in normal and pathophysiological conditions. Ann N Y Acad Sc 1963; 106:698-708. [PubMed]

Figure 4: Dr Giulio Barbero

The authors state - “Data relating to the tracheobronchial secretions and submaxillary saliva in cystic fibrosis indicate the high concentrations of various organic constituents which are secreted in this disease and the possible interrelationships of electrolytes on the physical properties of such constituents”.
So, as with most of these studies on the physico-chemical characteristics of the airway secretions, there were no firm conclusions which significantly advanced understanding of the basic defect of the condition. It is interesting that these authors again mentioned the possible role of the electrolyte content of the sputum which eventually turned out to be very relevant.

Professor Giulio Barbero (figure 4) was internationally recognised for his research in CF and patient advocacy. He published extensively on CF and general paediatric topics from 1953 to 2001. He was Head of Pediatrics at the University of Missouri, Columbia from 1972-1989. He gave the third Joseph Levy Memorial Lecture at the European CF Meeting in Jerusalem in 1996 entitled "The Science and Humanity of Cystic fibrosis".

1963 Kwart H, Mosley WW Jr, Katz M. The chemical characterization of human tracheobronchial secretion: a possible clue to the origin of fibrocystic mucus. Ann N Y Acad Sci 1963; 106:709-21. [PubMed]
One of many studies that attempted to show differences between normal and CF mucus. The authors summarise that their findings appear to support the basis for the disease they had advanced earlier i.e. that the extraordinary viscidity of CF mucus was formed as a result of a defect in “membrane chemistry”. They consider that the inference is consistent with the difficulties that CF individuals experience conserving sodium chloride.
So another group whose conclusions were not too far off the mark as it turned out. However, in contrast to the excessive loss of salt via the sweat people with CF had a deficiency of salt in the airway secretions due to the excessive absorption of sodium and reduced passage of chloride into the airways.

1963 Matthews LW, Spector S, Lemm J, Potter JL. Studies on pulmonary secretions I. The over-all chemical composition of pulmonary secretions from patients with Cystic Fibrosis, Bronchiectasis and Laryngectomy. Am Rev Resp Dis 1963; 88:199-204. [PubMed]
Leroy Matthews was one of the leading clinical authorities on CF in the USA at the time. He was developing his Comprehensive Treatment Programme in Cleveland which was to become the model for CF care adopted by the CF Foundation for their Centres programme. In this study they found that total solids, DNA protein, lipid and carbohydrate were highest in CF secretions, intermediate in non-CF bronchiectasis and lowest in normals.
The marked decrease in sodium and chloride in CF sputum which they found they considered was possibly related to the infection – “if this is so it makes untenable the hypothesis that the lowered sodium and chloride content of the cystic fibrosis secretions is etiologically responsible for the disease”.
This is interesting for later the “low salt theory” was to be regarded as the most likely explanation for the alteration in the pulmonary secretions and the persisting pulmonary infection problems.

1967 Denton R, Kwart H, Litt M. N-acetylcysteine in cystic fibrosis. Mechanical and chemical factors in treatment by aerosol. Am Rev Resp Dis 1967; 95:643-651. [PubMed]
Robert Denton describes the in vitro mucolytic activity, the clinical response and the effect of oxygen and nebulisation on the stability of a 20% solution of N-acetylcysteine. In vitro dispersion is more complete than with saline. There was an improvement in drainage from the upper respiratory tract but failure to improve drainage from the lower respiratory tract. Nebulisation did not alter the chemical structure. The present method of mask administration may not affect the lower respiratory tract (also Webb 1962 above; Reas 1963 above)

It is interesting that N-acetylcysteine is so rarely used for people with CF in the UK when some of the earlier published work is so impressive with regard to its mucolytic effect. More recently the relation to glutathione has caused renewed interest in the drug which when given orally in high doses provides a source of glutathione and apparently reduces airway inflammation (Tirouvanziam R, et al. Proc Nat Acad Sc 2006; 103:4628-4633).

1975 Wood RE, Wanner A, Hirsch J, Farrell PM. Tracheal mucociliary transport in patients with cystic fibrosis and its stimulation by terbutaline. Am Rev Respir Dis 1975; 111:733-8. [PubMed]
Robert Wood observed the movement of Teflon discs passing up the trachea through a bronchoscope in 14 adults with cystic fibrosis. The discs moved at 2.6 mm/min in CF and 20.1 mm/min in controls; terbutaline increased the speed of movement in CF to 5.5mm/min. The authors speculated, correctly as it has now become apparent, that this “may play a role in the pathogenesis of pulmonary disease”.
Robert Wood pioneered fibreoptic bronchoscopy in children with CF. The 3.5mm paediatric version of the bronchoscope he used in 1975 had no suction port which he circumvented by attaching a fine Teflon tube to the side allowing its use down to children aged 18 months; it was developed by the Olympus Corporation into the first flexible pediatric bronchoscope in 1978 at Dr Wood’s request.

1990 Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase 1 reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci 1990; 87:9188-9192. [PubMed]
An early report of the significant effect of recombinant human DNase1 (Pulmozyme) on sputum viscosity in people with CF by Steve Shak (Figure 17). To evaluate the potential clinical utility of recombinant human DNase I (rhDNase) in the treatment of CF, the authors cloned, sequenced, and expressed rhDNase. Catalytic amounts of rhDNase greatly reduced the viscosity of purulent CF sputum, transforming it within minutes from a non-flowing viscous gel to a flowing liquid. The reduction in viscosity is associated with a decrease in size of the DNA in the sputum. The authors suggested that inhalation of an rhDNase aerosol may be a simple direct approach that would help individuals with CF, and other patients with pneumonia or bronchitis, to clear their airways of purulent secretions.

In 1950 Armstrong JB & White JC (Lancet 1950; 2:739-742) had shown that bovine pancreatic DNase1, added to viscid purulent sputum, destroyed the extra cellular fibres of DNA and reduced the viscosity. Later Elmes PC & Armstrong JB (Thorax 1953; 8:295-300) reported its use in chronic bronchitis, but the side effects of the bovine preparation eventually precluded its use and further development at that time (Raskin P. Am Rev Respir Dis 1968; 98:697-698). Subsequently the rhDNase (Pulmozyme) reported here proved to be one of, if not the, major therapeutic advance of the Nineties. By 2005 Pulmozyme was taken by almost 70% of people on the US CF Foundation patient registry; eventually it would be used earlier and by milder affected and younger patients.

Figure 17: Dr Steve Shak. From New Insights into Cystic fibrosis. September 1993.

1950 Armstrong JB & White JC (Lancet 1950; 2:739-742. [PubMed]) had shown that bovine pancreatic DNase1, added to viscid purulent sputum, destroyed the extra cellular fibres of DNA and reduced the viscosity. Later Elmes PC & Armstrong JB (Thorax 1953; 8:295-300.[PubMed] ) reported its use in chronic bronchitis, but the side effects of the bovine preparation eventually precluded its use and further development at that time (Raskin P. Am Rev Respir Dis 1968; 98:697-698.[PubMed] ). Subsequently the rhDNase (Pulmozyme) reported here proved to be one of, if not the, major therapeutic advance of the Nineties. By 2005 Pulmozyme was taken by almost 70% of people on the US CF Foundation patient registry; eventually it would be used earlier and by milder affected and younger patients.

1993 Ranasinha C, Assoufi B, Shak S, Christiansen D, Fuchs H, Empey D, Geddes D, Hodson M. Efficacy and safety of short-term administration of aerosolised recombinant human DNase I in adults with stable stage cystic fibrosis. Lancet 1993; 342:199-202. [PubMed]
A phase II trial of DNase (Pulmozyme) from the Brompton Hospital, London, in which the mean percentage change in FEV1 from baseline was a 13.3% rise on rhDNase and only a 0.2% fall on placebo (p < 0.001). There was an insignificant rise of FVC of 7.2% in the rhDNase group and 2.3% in the placebo group. This study provided further confirmation that short-term administration of rhDNase in stable patients with cystic fibrosis was safe and resulted in an impressive improvement in lung function (also Shak et al, 1990 above; Fuchs et al. 1994 below).

1994 Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash ML, Ramsey BW, Rosenstein BJ, Smith AL, Wohl ME. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group. N Engl J Med 1994; 331:637-642. [PubMed]

Figure 29.1 Dr Henry J Fuchs. From www.bmm.com

Dr Henry J Fuchs, while working at Genentech from 1987 to 1996, led the clinical programme that resulted in the approval of Pulmozyme - one of the major clinical advances introduced during the Nineties. Dr Fuchs is now Senior Vice President and Chief Medical Officer of Biomarin. This report, the main publication on rhDNase (Pulmozyme) describes a randomized, double-blind, placebo- controlled study to determine the effects of once-daily and twice- daily administration of rhDNase on frequency of exacerbations of respiratory symptoms requiring parenteral antibiotics and on pulmonary function. A total of 968 adults and children with cystic fibrosis were treated for 24 weeks as outpatients. As compared with placebo, the administration of rhDNase once daily and twice daily reduced the age-adjusted risk of respiratory exacerbations by 28 percent (P = 0.04) and 37 percent (P < 0.01), respectively. The administration of rhDNase once daily and twice daily improved forced expiratory volume in one second during the study by a mean (+/- SD) of 5.8 +/- 0.7 and 5.6 +/- 0.7 percent, respectively. Transient voice alteration and laryngitis were more frequent in the rhDNase-treated patients.

This was the definitive Phase III trial showing the significant effect of inhaled rhDNase – one of the most important large multicentre studies of the 1990s. Pulmozyme was undoubtedly one of the major clinical advances of the decade and was licensed by the FDA in 1994 following this study. Regular treatment with DNase would become widely used (by almost 70% of people with CF on the CFF registry by 2005) first amongst those with significant chest involvement and then later for those with milder chest problems (Quann et al, 2001 below) and eventually, in some CF units for infants. The cost (approximately £7000 per year) proved a problem in certain parts of the UK. (Also Shak et al, 1990 above; Ranasinha et al, 1993 above)

1996 Robinson M, Regnis JA, Bailey DL, King M, Bautovich GJ, Bye PTP. Effect of hypertonic saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am J Respir Crit Care Med 1996; 153:1503-1509. [PubMed]
After inhalation of hypertonic (7%) saline alone, and with amiloride, the amount of radio aerosol cleared from the right lung at 60 and 90 minutes was significantly increased. The authors suggested that inhaled hypertonic saline was a potentially useful treatment for CF.
Peter Bye’s group at St Vincent’s Hospital in Sydney continued their work on hypertonic saline and eventually carried out a successful clinical trial of hypertonic saline in adults with CF and confirmed the value of hypertonic saline treatment (Elkins et al, 2006 below).
Hypertonic 7% saline inhalations were originally reported to double the rate of removal of bronchial secretions in chronic bronchitis in an interesting study using radio aerosols (Pavia et al, Am Rev Respir Dis 1978; 117:199-203). I recall consulting Margaret Hodson in the early Eighties on any suggestions for treating a girl with CF who had excessively viscid secretions. She advised a trial of nebulised hypertonic saline which, from her personal experience, had proved helpful.

2000 Pons G, Marchand MC, d'Athis P, Sauvage E, Foucard C, Chaumet-Riffaud P. The Amiloride-AFLM Collaborative Study Group. Pediatr Pulmonol 2000; 30:25-31. [PubMed]
In this large French study 64 patients with CF, chronically infected with Pseudomonas aeruginosa, received either nebulised amiloride or placebo three times daily for 6 months in addition to their usual treatments. The study failed to demonstrate any significant benefit from the adding the amiloride to their treatment.

This multicentre French study confirmed the lack of significant clinical effect of inhaled amiloride the use of which had been of interest as a treatment since Michael Knowles's amiloride study in the early Nineties - using amiloride to reduce the excessive sodium absorption from the CF airways (Knowles MR et al. N Eng J Med 1990; 322:1189-1194. [PubMed] above). Apparently the usual preparation of amiloride is rapidly destroyed in vivo. Excessive Na absorption in now considered important in the pathophysiology of the respiratory tract, other ways of inhibiting excessive sodium absorption have replaced amiloride as treatment possibilities. Longer acting analogues of amiloride may be more effective and subsequently became available such as compound 552-02 (Hirsch AJ et al. J Pharmacol Exp Ther 2008; 325:77-88. [PubMed] above in Science section). Subsequent development of the compound by Gilead has continued as Gilead GS9411 which has entered clinical trials in 2009.

2001 Suri R, Metcalfe C, Lees B, Grieve R, Flather M, Normand C, Thompson S, Bush A, Wallis C. 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. [PubMed]
In an open cross-over trial, 48 children were allocated in random order to 12 weeks of once-daily rhDNase (2.5 mg), alternate-day rhDNase (2.5 mg), and twice-daily 5 ml 7% hypertonic saline. The primary outcome was FEV1. The mean FEV1 increased by 16% (SD 25%), 14% (22%), and 3% (21%) with daily rhDNase, alternate-day rhDNase, and hypertonic saline, respectively. There was no difference between daily and alternate-day rhDNase. However, daily rhDNase showed a significantly greater increase in FEV1 than hypertonic saline (8% {2 to 14}, p=0.01).

It is no surprise that hypertonic saline, delivered by jet nebuliser, was not as effective as daily rhDNase, although there was variation in individual response. Interestingly there was no evidence of a difference between daily and alternate-day rhDNase. Although inhaled hypertonic saline was less effective as a mucolytic THAN rhDNase, it was considerably cheaper. It must be stressed that these are group responses and there is considerable individual variation between patients and it is important that clinicians assess the individual response of each patient when prescribing these treatments. Also it was later shown failure to respond to rhDNase may be related to the magnesium status of the patient (Sanders NN et al, Thorax 2006; 61:962-968). [PubMed]

2001 Quan JM, Tiddens HAWM, Sy JP, McKenzie SG, Montgomery MD, Robinson PJ, Wohl ME, Konstan MW. Pulmozyme Early Intervention Trial Study Group. A two-year randomised placebo controlled trial of dornase alfa in young patients with cystic fibrosis with mild lung function abnormalities. J Pediatr 2001; 139:813-820. [PubMed]
This was a 96-week, randomized, double-blind, placebo-controlled trial involving 49 CF centers and 474 children. Inclusion criteria were age six to 10 years and forced vital capacity > or = 85% predicted. At 96 weeks the treated group had maintained their respiratory function levels; the treatment benefit for dornase alfa compared with placebo in percent predicted (mean +/- SE) was 3.2 +/- 1.2 for FEV1 (P =.006), 7.9 +/- 2.3 for FEF 25-75 % (P =.0008), and 0.7 +/- 1.0 for FVC (P =.51). The risk of respiratory tract exacerbation was reduced by 34% in patients who received dornase alfa. The authors concluded that treatment of young patients with CF with dornase alfa maintains lung function and reduces the risk of exacerbations over a 96-week period.
The results of this trial influenced some paediatricians to try young CF patients on Pulmozyme before they developed chronic infection and this became routine practice in at least one large UK Paediatric CF Centre and also in Copenhagen where the treatment was found to reduce the frequency of respiratory exacerbations and new positive respiratory tract cultures (Frederiksen et al, 2006 below).

2003 Berge MT. Wiel E. Tiddens HA. Merkus PJ. Hop WC. de Jongste JC. DNase in stable cystic fibrosis infants: a pilot study. J Cyst Fibros 2003; 2:183-188. [PubMed]
The authors treated nine CF patients (0.7-1.9 years) with nebulised rhDNase (2.5 mg) and NaCl 0.9% (10 ml) via jet nebulizer cross-over once daily during 2-week treatment blocks. Measurements were performed at baseline and after treatment blocks and consisted of lung function tests (plethysmography and tidal rapid thoraco-abdominal compression technique), overnight pulse oximetry, and daily symptom scores. The DNase treatment and the different assessments were well tolerated by all children and their parents. Lung function showed increased airway patency after treatment with rhDNase (P < 0.001), but not after NaCl 0.9%. This pilot study indicates that objective assessment of the effects of rhDNase is feasible in infants with CF who have little or no respiratory symptoms. Our results warrant a larger randomized placebo-controlled trial.

This small but detailed study from an expert unit showed definite short term benefit from inhaled rhDNase even in infants.At least one large paediatric centre in the UK tries CF infants on rhDNase as soon as they are able to inhale it.

2005 Fitzgerald DA. Hilton J. Jepson B. Smith L. A crossover, randomized, controlled trial of dornase alfa before versus after physiotherapy in cystic fibrosis. Pediatrics 2005; 116:e549-554. [PubMed]
Dornase alfa is equally efficacious when delivered before or after physiotherapy/PEP in patients with CF. Patients who are colonized persistently with P aeruginosa may derive more improvement in FEV1 when dornase alfa is delivered after physiotherapy/PEP.

2005 Robinson TE. Goris ML. Zhu HJ. Chen X. Bhise P. Sheikh F. Moss RB. Dornase alfa reduces air trapping in children with mild cystic fibrosis lung disease: a quantitative analysis. Chest 2005; 128:2327-35. [PubMed]
Twenty-five children with CF randomized to either daily rhDNase or placebo aerosol were evaluated at baseline, and at 3 months and 12 months by spirometer-triggered HRCT and spirometry. After 3 months of treatment, both groups had improvements in percentage of predicted FEV1 and FEF(25-75%), and total HRCT visual scores. In contrast, the rhDNase group had a 13% decrease in quantitative air trapping from baseline (severe air trapping [A3]), compared to an increase of 48% in the placebo group (p = 0.023). Quantitative air trapping (A3) remained improved in the rhDNase group (- 15.4%) and worsened in the placebo group (+61.3%) with nearly significant differences noted between groups (p = 0.053) after 12 months of treatment. CONCLUSIONS: Quantitative air trapping is a more consistent sensitive outcome measure than either spirometry or total HRCT scores, and can discriminate differences in treatment effects in children with minimal CF lung disease.

One of the earliest signs of CF is air trapping and over inflation on the chest X-ray. it would be reasonable to expect this to improve with rhDNase treatment. Indeed many paediatricians are now trying young children on rhDNase as soon as they are able to take it.

2006 Frederiksen B, Pressler T, Hansen A, Koch C, Hoiby N. Effect of aerosolized rhDNase (Pulmozyme) on pulmonary colonisation in patients with cystic fibrosis. Acta Paediatrica 2006; 95:1070-1074. [PubMed]
Patients received daily Pulmozyme or no Pulmozyme for 1 year. Positive cultures were obtained in 72% of treated and 82% of untreated patients. S. aureus was presented in 16% treated and in 30% of untreated. FEV1 increased by 7.3% in treated compared to only 0.9% of untreated group.

This was a useful observational study which, although the results were modest, provided practical valuable information that long term Pulmozyme appeared to reduce the need for antibiotics to eradicate new infections and also improved the lung function of patients who were not chronically infected.

2006 Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT. National Hypertonic Saline in Cystic Fibrosis Study Group. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Eng J Med 2006; 354:229-240. [PubMed]
Inhaled hypertonic saline (7%) acutely increases mucociliary clearance and, in short-term trials, improves lung function in people with cystic fibrosis. Peter Bye (figure 40)  of St Vincent’s Hospital Sydney, and Mark Elkins (figure 41) and their colleagues tested the safety and efficacy of inhaled hypertonic saline after a bronchodilator in a long-term double-blind, parallel-group trial over 48 weeks. The rate of change (slope) in lung function did not differ significantly between the treated and control groups but the absolute difference in lung function between groups was significant (P=0.03) when averaged across all post-randomization visits. The hypertonic-saline group also had a 56% reduction in pulmonary exacerbations. This study was funded by the US CF Foundation and received considerable attention.

A similar short term study from Chapel Hill, USA showed similar benefit in mucus clearance and FEV1 (6.62% increase) but not when used with amiloride which was considered to inhibit osmotically driven water transport (Donaldson SH et al. NEJM 2006; 354:241-250. [PubMed]).
Subsequently 7% saline was shown to be tolerated by infants with CF when assessed by infant respiratory function tests and clinically (Subbarao P et al, Pediatr Pulmonol 2007; 42:471-476. [PubMed]) and will be subjected to a multicentre trial funded by the CF Foundation.

The great interest in these findings stemmed partially from the fact that here was a way of correcting the low salt situation, postulated by Boucher to be a major cause of the fluid deficit and airway mucociliary clearance problems in CF. Also the treatment is inexpensive compared with other very expensive mucolytics such as rhDNase.

2006 Tirouvanziam R. Conrad CK. Bottiglieri T. Herzenberg LA. Moss RB. Herzenberg LA. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. Proc Nat Acad Sci USA 2006; 103(12):4628-33. [PubMed]
Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment. After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

Conflicting views on the effect of N-acetylcysteine appear despite the Cochrane conclusion that it is of no benefit in CF.

2006 Sanders NN, Franckx H, De Boeck K, Haustraete J, De Smedt SC, Demeester J. Role of magnesium in the failure of rhDNase therapy in patients with cystic fibrosis. Thorax 2006; 61:962-968. [PubMed]
rhDNase (Pulmozyme) is a major component of treatment for people with CF but some patients show no or little response and hence fail to benefit from this important drug. In this study the biochemical properties, physical properties, and degradation by rhDNase-I of sputum obtained from clinical responders and non-responders were compared; also the ability of magnesium to reactivate rhDNase-I in DNA solutions and in sputum was investigated.
The effect of oral magnesium supplements on magnesium levels in the sputum of patients with CF was also examined. Sputum from clinical responders was extensively degraded in vitro on incubation with rhDNase-I, while sputum from clinical non-responders was not degraded: the median decrease in sputum elasticity in the two groups was 32% and 5%, respectively.
Sputum from clinical responders contained significantly higher concentrations of magnesium than sputum from non-responders (2.0 mM v 1.3 mM; p = 0.020). Sputum that could not be degraded by rhDNase-I became degradable after preincubation with magnesium. The effect of magnesium on rhDNase-I activity was mediated through actin. Oral intake of magnesium enhanced the magnesium concentration in the sputum of CF patients. So increasing the magnesium concentration in sputum by, for example, oral magnesium supplements may be a promising strategy to overcome the failure of rhDNase-I in some patients with CF.

One further supportive study by Rosenecker J et al. (Airway surface liquid contains endogenous DNase activity which can be activated by exogenous magnesium. Eur J Med Res 2009; 14:304-308. [PubMed]). They found BAL samples degraded plasmid DNA only after pre-incubation with magnesium. When analyzing the exhaled breath condensate the samples obtained from the healthy volunteers showed no DNase activity even after pre incubation with magnesium, whereas in one of the two samples obtained from CF patients contained DNase activity after pre-incubation with magnesium. The authors concluded that Increasing the magnesium concentration in the airway surface liquid by aerosolisation of magnesium solutions or oral magnesium supplements could improve the removal of highly viscous mucus in chronic lung disease by activating endogenous DNase activity.

Fig. 44: Professor Kris De Boeck

Professor Kris De Boeck (figure 44) of University Hospital Leuven, Belgium is a leading, very active researcher and clinician in the CF field. Potentially a very useful paper which could benefit the 30-50% of patients who fail to show a significant response to rhDNase.

2006 Frederiksen B, Pressler T, Hansen A, Koch C, Hoiby N. Effect of aerosolized rhDNase (Pulmozyme) on pulmonary colonization in patients withScystic fibrosis. Acta Paediatrica 2006; 95:1070-1074. [PubMed]
During one year patients with CF were randomized either to aerosolized Pulmozyme daily or to no Pulmozyme. The number of positive respiratory cultures was higher in the untreated group (82%) compared with the treated group (72%) (p<0.05). The most striking difference was found for Staphylococcus aureus, with a prevalence of 30% in the untreated group compared with 16% in the treated group (p<0.0001). Pulmonary function (FEV1) in the treated group showed a significant increase of 7.3% compared to 0.9% in the untreated group (p<0.05). Long term DNase treatment was beneficial to CF patients without chronic lower respiratory tract infection, with fewer positive respiratory cultures, leading to reduced demand for antibiotics and to improved lung function.

A useful study from Copenhagen showing inhaled rhDNase reduced the likelihood of a positive respiratory culture. This finding provides additional support for starting all patients on the treatment at an early age, where inhaled treatment is practical, as occurs now in some centres.

2007 Broughton-Head VJ. Shur J. Carroll MP. Smith JR. Shute JK. Unfractionated heparin reduces the elasticity of sputum from patients with cystic fibrosis. Am J Physiol - Lung C 2007; 293:L1240-9. [PubMed]
Unfractionated heparin significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5 +/- 2.5% at baseline to 36.9 +/- 4.4% at 10 mg/ml but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.

There were subsequent studies from Southampton and Portsmouth in the UK although an earlier pilot study using inhaled heparin 50,000 IU twice daily showed no clinical effect - a larger dose was suggested. [PubMed]

2007 McKenzie SG, Chowdhury S, Strandvik B, Hodson ME. Investigators of the Epidemiologic Registry of Cystic Fibrosis. Dornase alfa is well tolerated: data from the epidemiologic registry of cystic fibrosis. Pediatr Pulmonol 2007; 42:928-937. [PubMed]
After closure of the Epidemiologic Registry of Cystic Fibrosis, a comprehensive safety analysis of dornase alfa (Pulmozyme) was performed. The results confirm the safety of dornase alfa in people with CF of all ages. Children under 5 years old appear to tolerate dornase alfa at least as well as older patients.

There is an accumulating literature on the use of rhDNase in people with CF and most is favourable, confirming its beneficial effect and safety. In one large CF unit in the UK all children are given a trial on rhDNase as soon as inhalation therapy becomes feasible. Undoubtedly rhDNase is one of the more important treatment advances of the past 15 years (Shak et al, 1990 above; Fuchs et al, 1994 above).

Fig. 49: Dr Sheila McKenzie

Dr Sheila McKenzie of Hoffmann-La Roche ltd, has been involved with the development and introduction of Pulmozyme since the first clinical trials in the Nineties shortly after Pulmozyme was licensed. She was involved in the latter stages of the European phase IIIb study (published in 1998), the implementation (but not the original planning) of ERCF and the planning & implementation of the Quan mild patient study. She is now (in 2010) the longest-serving member of Roche & Genentech staff to have worked with CF.

2007 Boucher RC. Evidence for airway surface dehydration as the initiating event in airway disease. J Int Med 2007; 261:5-16. [PubMed]
A very clear review marshalling the relevant current experimental and clinical evidence for Richard Boucher’s “too little salt” theory. Airway surface dehydration due to excessive sodium absorption and reduced chloride secretion is the main cause of the CF lung being unable to defend itself against infection. The hypothesis predicts the airway surface dehydration produces the mucus adhesions, inflammation and bacterial biofilm formation characteristic of cystic fibrosis.

Fig. 48: Professor Richard Boucher (From www.med.unc,edu website)

Richard Boucher (Figure: from NACFC conference) is Professor and Director Cystic Fibrosis/Pulmonary research Centre at the University of North Carolina, Chapel Hill has for many years been a leader in CF research and long time protagonist of the “low salt theory”.
The theory is convincing. The development of lung disease in transgenic ENaC-over expressing mouse models has been convincing suggesting that the entire early spectrum of CF-like lung disease is related to dehydration in the airways. Inhaled hypertonic saline, drawing water into the airways, has shown significant clinical benefit in people with CF. Also INS37217 – a metabolically stabilised form of UTP – has shown beneficial effects and is entering Phase III trials; also a long acting third generation descendant of the sodium channel blocker amiloride is in Phase II trials.

2007 Subbarao P. Balkovec S. Solomon M. Ratjen F. Pilot study of safety and tolerability of inhaled hypertonic saline in infants with cystic fibrosis. Pediatr Pulmonol 2007; 42:471-476. [PubMed]
A prospective trial of inhaled HS in infants with CF. Raised volume rapid thoracoabdominal compression (RVRTC) maneuvers were performed at baseline, 10 min after salbutamol inhalation and 15 min after inhalation of a 7% HS solution. Oxygen saturation, respiratory rate, heart rate, and cough frequency were recorded during each inhalation. A clinically important change in lung function was defined a priori as a change in FEV 0.5 of > or =20%. Thirteen infants (5 female) aged 25-140 weeks were enrolled in the study. Overall, there was no difference between FEV(0.5) or FEF(25-75) at baseline, after bronchodilator or after HS. Respiratory and heart rate as well as oxygen saturation remained stable during inhalation of the HS. Three infants had cough during inhalation; one of the infants woke up due to cough but recovered within 5 min. No other side effects were observed during or immediately after inhalation. There was no difference in microbiologic yield between pre- and post-HS throat swabs. In this pilot study, inhalation of HS was well tolerated in CF infants. These results support a study of the efficacy of HS in this age group - a trial was planned and is in progress (2011).

2008 McLaughlin AM. McGrath E. Barry R. Egan JJ. Gallagher CG. Treatment of lobar atelectasis with bronchoscopically administered recombinant human deoxyribonuclease in cystic fibrosis? Clin Respir J 2008; 2:123-126. [PubMed]
The objective of this study was to describe our experience in which rhDNase (Pulmozyme) was administered by bronchoscopic instillation into atelectatic lobes in five adults with CF. This method was successful in treating lobar atelectasis, which was resistant to conventional therapy with antibiotics and physiotherapy. In all but one of the cases we described, administration of DNase in this manner resulted in a radiographic and clinical improvement of the atelectasis. We recommend that respiratory physicians consider this as a second line treatment in the management of atelectasis.

Although not the first to use this treatment for atelectasis, confirmation of the success of this treatment is useful for clinicians faced with resistant atelectasis.

2008 Jaques A. Daviskas E. Turton JA. McKay K. Cooper P. Stirling RG. Robertson CF. Bye PT. Lesouëf PN. Shadbolt B. Anderson SD. Charlton B. Inhaled mannitol improves lung function in cystic fibrosis. Chest 2008; 133:1388-1396. [PubMed]
Inhaled mannitol is an osmotic agent that increases the water content of the airway surface liquid, and improves the clearance of mucus with the potential to improve lung function and respiratory health. To this end, this study examined the efficacy and safety of therapy with inhaled mannitol over a 2-week period in a randomized, double-blind, placebo-controlled, crossover study. Thirty-nine subjects with mild-to-moderate CF lung disease inhaled 420 mg of mannitol or placebo twice daily for 2 weeks. Following a 2-week washout period, subjects were entered in the reciprocal treatment arm. Lung function, respiratory symptoms, quality of life, and safety were assessed. Mannitol treatment increased FEV(1) from baseline by a mean of 7.0% (95% confidence interval [CI], 3.3 to 10.7) compared to placebo 0.3% (95% CI, - 3.4 to 4.0; p < 0.001). The absolute improvement with mannitol therapy was 121 mL (95% CI, 56.3 to 185.7), which was significantly more than that with placebo (0 mL; 95% CI, - 64.7 to 64.7). The forced expiratory flow in the middle half of the FVC increased by 15.5% (95% CI, - 6.5 to 24.6) compared to that with placebo (increase, 0.7%; 95% CI, - 8.3 to 9.7; p < 0.02). The safety profile of mannitol was adequate, and no serious adverse events related to treatment were observed. Inhaled mannitol treatment over a period of 2 weeks significantly improved lung function in patients with CF. Mannitol therapy was safe and well tolerated.

Mannitol was developed further and licensed in 2010.

2008 Padman R. Werk LN. Ramirez-Garnica G. Ye G. Nathanson I. Association between practice patterns and body mass index percentile in infants and young children with cystic fibrosis. J Cyst Fibros 2008; 7:385-390.[PubMed].
Data from the CF Registry on 165 infant who were using Pulmozyme before the age of 2 years suggested that this early use of dornase alpha may improve nutritional outcome through age six.

2009 Dauletbaev N. Fischer P. Aulbach B. Gross J. Kusche W. Thyroff-Friesinger U. Wagner TO. Bargon J. A phase II study on safety and efficacy of high-dose N-acetylcysteine in patients with cystic fibrosis. Euro J Med Res 2009; 14:352-358. [PubMed]
A single-centre, randomised, double-blinded, placebo-controlled phase II clinical study to test safety and efficacy of a 12-week therapy with low-dose (700 mg/daily) or high-dose (2800 mg/daily) of NAC. High-dose NAC was a well-tolerated and safe medication but did not alter clinical or inflammatory parameters. However, extracellular glutathione in induced sputum tended to increase on high-dose NAC. The authors concluded that high-dose NAC is a well-tolerated and safe medication for a prolonged therapy of patients with CF with a potential to increase extracellular glutathione in CF airways.

This study confirms the lack of clinical effect of N-acetylcysteine on the respiratory function and inflammatory parameters but the increase in extracellular glutathione may be of some benefit. It is difficult to reconcile the lack of clinical effect with some of the early reports showing marked increase in the volume of sputum; also the clinical experience suggesting some benefit. The effects are complicated - even recently it has been reported that that NAC causes a significant efflux of Cl from CF bronchial epithelial cells (Veralogiani G et al. [PubMed])

2009 Rosenecker J. Naundorf S. Rudolph C. Airway surface liquid contains endogenous DNase activity which can be activated by exogenous magnesium. Eur J Med Res 2009; 14:304-308. [PubMed].

Increasing the magnesium concentration in the airway surface liquid by aerosolisation of magnesium solutions or oral magnesium supplements could improve the removal of highly viscous mucus in chronic lung disease by activating endogenous DNase activity

An interesting observation as magnesium had already been involved in failure of DNase therapy in people with CF (Sanders NN et al. 2006.[PubMed].

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