British Medical Bmllttix (1992) NW 48, No 4, pp7S5-«M O The Bntuh Council 1992

New treatments for cystic fibrosis E Alton, N Caplen, D Geddes, R Williamson Royal Brompton & St Mary's Hospital Medical School, London, UK

The cornerstones of CF medical treatment remain optimised nutrition, antibiotics and chest physiotherapy. There are however a number of promising new approaches which may add considerably to these traditional treatments. The most fundamental prospective new treatment is gene therapy which is still a long way off but could, in theory, provide a virtual cure for the disease. Less visionary but potentially as important is drug therapy aimed at the basic defect in airway ion transport. Both of these approaches are best suited to patients with early or even pre-symptomatic disease. In contrast, a third group of new treatments may prove useful in patents with established lung damage; these include a variety of anti-inflammatory drugs and DNase. This chapter will discuss each of these new treatments as well as exploring the difficult issue of assessing new treatments in pre-symptomatic disease.

GENE THERAPY

The identification and cloning of the cystic fibrosis (CF) gene and the description of the protein for which it codes, the cystic fibrosis transmembrane conductance regulator (CFTR) has opened the way for the development of a treatment for cystic fibrosis based on the transfer of a normal copy of the CF gene into pathologically affected cells.1"3 The prospects for gene therapy have been significantly improved as a result of two independent sets of experiments. These demonstrated that the introduction of a single copy of the normal cDNA corresponding to the coding sequence of the CF gene into cells carrying the CF defect in vitro restored normal cAMP dependent chloride conductance.4.5 The logical extension of these studies is that if the CF gene can be delivered to a sufficient number of cells in vivo and normal protein function established in them, the clinical symptoms of CF should be alleviated. Here the

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recent progress made in developing methods for transferring genes into cells will be reviewed. These are the first essential prerequisite for the in vivo introduction of the CF gene. It is likely that most early attempts at gene therapy will concentrate on targeting of the lung epithelium. However, in the future treatments of pancreatic and liver dysfunction may also be possible. DNA transfer Present techniques used routinely in the laboratory to transfer DNA into mammalian cells in tissue culture fall into three main classes: those that physically introduce DNA into cells directly; those that exploit chemical interactions between DNA:ligand conjugates and the cell surface membrane and those that require the use of biological intermediates, such as viruses. Physical methods Examples of methods that physically transfer DNA into cells are electroporation and micro-injectionA7 Both of these methods give a high efficiency of transfection. Electroporation, however, results in significant cell death and micro-injection is only possible on a limited number of cells. It may eventually be possible to transfect sufficient respiratory epithelium progenitor cells by micro-injection in vitro and then to regraft the lung with corrected cells. This, however, would be an excessively labour intensive process, and thus an unlikely approach for the targeting of this tissue. Theoretically micro-injection of DNA into fertilized human eggs is a potential approach to gene therapy but has no real application for CF and in any case treatments leading to germ line conversion are controversial and may not be legally permitted. Liposomes Liposomes are artificial cationic lipid vesicles that spontaneously bind DNA and fuse to cell membranes and have been used to transfer several genes into cells in tissue culture.8-9 The system gives a high efficiency of transfer but with little or no cytotoxic effect and can be used for both the generation of transient and stable cell transfectants in vitro. Given the extensive study of aerosol delivery systems for other compounds, such as antibiotics, to patients with cystic fibrosis it may well be possible to adapt this technology for the delivery of DNA bound by liposomes. Liposomes have the added advantage that other molecules can be conjugated to the lipids. Such molecular conjugates can be used

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to increase the efficiency at which the target cell is transfected. For example, a monoclonal antibody can be incorporated into the lipid bilayer of the liposome, so directing the liposome to a specific target.10-11 Receptor mediated endocytosis By linking various receptor specific ligands to polycations such as protamine or poly-lysine that bind nucleic acid, DNA has been delivered to avian erythroblasts and human leukemic cells via the transferrin receptor12-13 and to liver cells via the asialoglycoprotein receptor.14 Recently Curiel and co-workers15 have reported the successful uptake of the luciferase reporter gene via the transferrin receptor by a cystic fibrosis respiratory epithelium cell line, CFT1. This strategy has an advantage in comparison with other methods in its potential for specific targeting to different cell types depending on the receptor-ligand combination used. It should also be safe with the possibility of being able to introduce DNA repeatably by this means. Viral vectors Of the biologically based systems available for mediation of the gene transfer, retroviruses are at present most widely used. Most retroviral vectors are derived from the Moloney murine leukemia virus (MoMLV), a murine RNA virus which causes a T cell lymphocytic leukemia. The proviral DNA generated by reverse transcripton of the viral RNA is isolated and converted into a double-stranded DNA plasmid for use in gene delivery. This can then be modified and re-packaged in the presence of a helper virus to generate infectious viral particles. Many retroviral vectors have now been generated which vary in the elements altered or removed to ensure the vector is replication deficient and that wild-type virus cannot be reformed, as well as in the sequences used to control gene expression and the exogenous gene used. Retroviruses have been used for the successful introduction of many genes in vitro and are now being used in animals and humans. The adenosine deaminase (ADA) gene has been introduced into the bone marrow cells of primates via retrovinis mediated gene transfer16 and clinical trials of this system to treat severe combined immune-deficiency disease caused by ADA deficiency have now been initiated in the United States under the direction of the National Institute of Health, Bethesda, with early encouraging results (personal communication, Dr Paul Tolstoshev). The CFTR cDNA can be transferred to and expressed in CFPAC-1 cells, a pancreatic adenocarcinoma cell line derived from a CF patient, by retrovinis mediated transfer, and normal chloride transport

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established.4 In addition cell lines derived from these experiments have been reported to show normal chloride ion permeability for at least 180 days after gene transfer.17 However, there are several problems with the use of retroviruses in the treatment of cystic fibrosis. Retroviruses will only infect dividing cells18 and a large proportion of the cells likely to be targeted for correction of the CF defect are terminally differentiated. There is a risk of integration within potentially oncogenic sequences, or the disruption of other functionally important genes. This has been considered to be of minor risk in the transfer of ADA gene to children with SCID, but is likely to be of more importance in relation to the treatment of cystic fibrosis, because repeated administration in vivo is probably going to be required to obtain sufficient corrected cells to see a clinical effect. In addition, retroviral transfer is non-targeted and thus cells other than respiratory epithelium cells are likely to be transfected. A retrovirus vector has been used to transfer and express a large portion of the human CFTR cDNA in NTH 3T3 mouse fibroblast.19 These cells do not normally express the CF gene but once transfected the cells exhibit cAMP dependent chloride transport. Similar results have been found when other transfer systems have been used to introduce the CF gene into non-epithelial cells.20- 21 This would suggest that in vivo non-targeted introduction of the CF gene would result in the generation of CFTR dependent function in cells that do not normally express the CF gene. As yet we do not know what the clinical effect of overexpression and/or ubiquitous expression of the CF gene will be. Recently, transgenic mice have been generated that express high levels of the CFTR in pulmonary tissue as a result of linking the human CFTR cDNA to lung epithelial-specific promoters, with no obvious effect on the development of the animals.22 Additional work is still required to establish the long term effects of generalized CFTR function in the lung and other tissues. Given the inherent problems associated with using retroviruses as a means of gene transfer in vivo, particularly for the delivery of those tissues affected by CF, other viral-based systems are also being considered. Adenovims is a medium sized non-enveloped virus mainly associated with infection of the respiratory tract and the eye; there is a link between adenovims and the development of tumours in experimental animals, but there is no evidence for it being responsible for malignancy in humans. The adenovirus genome is a linear, double stranded DNA which can be modified to allow insertion of up to 7.5kb of exogenous DNA,23 which would accommodate the full length CFTR cDNA sequence and its various controlling elements. Some adenovirus subgroups are naturally trophic for respiratory lung epithelium and thus should target many of those cells requiring correction by the CF gene.

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An adenovirus based vector has been successfully used to transfer recombinant alpha-1-antitrypsin gene to lung epithelium of the cotton rat by instillation of the trachea24 with the production of the protein for at least one week. Similar experiments using the same adenovirus-based vector and the human CFTR cDNA have shown the presence of human CFTR transcripts for up to 6 weeks in the lungs of cotton rats treated in this way, with human CFTR protein being detected 11-14 days after infection.25 In addition CFPAC-1 cells infected with the recombinant adenovirus vector containing the normal CF gene showed corrected cAMP-mediated chloride permeability. Though the results of these experiments are encouraging the safety and efficacy of the system will need to be rigorously investigated before clinical trials can be considered. Recently, the CF gene has been transferred in vitro into human bronchial epithelial cells isolated from individuals with cystic fibrosis.26 Another possible viral delivery system is based on the single-stranded DNA virus, adeno-associated virus (AAV). Adeno-associated virus is a human parvovirus which requires a helper virus for infection, usually an adenovirus or herpes virus. In the absence of helper virus the AAV genome integrates into the host chromosomal DNA.27 The virus is naturally trophic for respiratory epithelium and though there is a significant size limitation to the DNA that can be packaged (5kb) the minimal CF coding sequence has been cloned into an AAV based vector.28 Preliminary experiments have shown that AAV transducing particles containing this construct can correct the CF defect in cultured cells29 and thus it may be possible to use AAV based vectors in vivo. All the above viral-based vector systems have significant disadvantages: a limitation of the size of foreign DNA that can be incorporated, a potential risk of tumour formation as a result of random integration within the host genome and the possibility of regeneration of wild-type virus. Artificial chromosomes The use of an episomally carried artificial chromosome containing the minimum elements required for stable segregation as well as the gene of interest would be an ideal construct to overcome many of the problems inherent in the use of virally based vectors. At present no mammalian artificial chromosome has been constructed. The limit to this technology is our lack of understanding of the structure and functional requirements of the mammalian centromere, although artificial chromosomes containing a yeast centromere and teleomeres have been developed. Yeast artificial chromosomes (YACs) were initially used as a tool for cloning

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large pieces (up to 1 megabase) of DNA.30 Experiments have been performed introducing a YAC into mammalian cells via polyethylene glycol-mediated fusion.3132 However, results showed that the DNA introduced by this manner is unstable unless integrated into the host chromosome, presumably because the yeast centromere will not allow normal segregation of the yeast chromosome at mitosis in a mammalian cell. 32 Despite these initial problems, there are good prospects for determining and altering those sequences required to stabilize existing YACs and generate new artificial chromosomes. This will undoubtedly be one of the long term approaches to gene therapy, although mechanisms to allow delivery of such large pieces of DNA intact will also have to be developed in parallel. Future directions While most research effort has so far been directed towards techniques of gene transfer and correction of the chloride channel defect in vitro, this is only the beginning. A number of key questions will need to be answered before gene therapy can be considered for clinical trials: 1. Which cells to target and how often? While it may be sufficient to target fully differentiated cells, this is not certain both because there are at least eight differentiated cell types in the airways and also because these cells turn over relatively quickly. It may be better to identify and target the epithelial stem cell in the hope of obtaining more durable expression of the inserted gene. Adenovirus transfer of antitrypsin into the cotton rat resulted in protein synthesis which was detectable for at least a week, but weekly treatment for life is a daunting prospect and less frequent treatment or ideally stable expression would be preferable. 2. Host defences Repeated administration of foreign material, whether viral vector or human DNA is likely to give rise to antibody formation. This may prevent or impair DNA transfer or alter its incorporation and subsequent expression. 3. Control of expression mRNA levels for CFTR are very low (1-2 copies/cell) in the airway epimelium. This suggests that only low levels of expression are required for normal function which may make adequate gene transfer easier to

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achieve. Conversely it raises the possibility of risk from over expression and it is likely that control elements will be needed as well as simply a copy of the normal gene. Safety studies could be particularly difficult to do as a minor disadvantage may take years to detect. All these problems and many more must be solved before clinical trials will be possible. To this end the availability of an animal model for cystic fibrosis will significantly increase our ability to test and assess the safety of many of the approaches to gene therapy outlined here and thus experiments to develop a transgenic mouse with a defective CF gene are now well under way33 (Dorin, Porteous, Wainwright & Halford, personal communication). ION TRANSPORT THERAPY FOR CYSTIC FIBROSIS Over the last decade it has been established that the basic defect in CF centres around abnormalities of ion transport. This section briefly reviews the evidence for this and the very recent beginnings of therapy aimed at re-establishing normal ion transport in these patients. The basic defect in CF centres around abnormal ion transport It has been recognized for several centuries that the presence of excess salt on the skin of infants indicates a poor prognosis.34 These observations were underlined by Di Sant 'Agnese in 195335 when during a heatwave in New York he correctly attributed the collapse of several CF children to an excessive loss of salt in their sweat. Since the formation of hypotonic sweat is dependent on the active reabsorption of sodium (with consequent passive absorption of chloride across sweat duct epithelial, these cells became a logical target for investigation. In a series of elegant studies Quinton demonstrated, both in vitro36 and in vivo37 that CF sweat ducts produce a markedly more negative potential difference (PD) across their surfaces. In addition, he showed that this raised PD related to impermeability of the ductal epithelium to chloride with secondary inhibition of sodium reabsorption. With respect to the lungs, ion transport in human airways is dominated by absorption of sodium from the mucosal surface (Fig. 1). Thus, the Na+/K+/ATPase in the basolateral surface of these cells creates a favourable gradient for the movement of sodium into cells through sodium channels located in the apical membrane. This movement is accompanied osmotically by water, probably principally through paracellular pathways. It is likely that human airways are also capable of secreting chloride into the airway lumen, although this process appears to be of lesser quantitative importance. Chloride enters the cell via

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MUCOSAL

N«

2CI

SEROSAL

Fig. 1 Principal ion movements across respiratory epithebal cells

a basolaterally-sited cotransporter taking advantage of the favourable electrochemical gradient for sodium entry. Consequently, a suitable gradient will exist for chloride exit through apically-sited channels. Again, water will follow such net transport. It is likely that the balance of these transport processes determines the volume and composition of airway surface liquid, which in turn may have an important role in regulating mucociliary clearance.38 In 1981, Knowles et al 39 demonstrated that the nasal and lower respiratory epithelium of CF patients had a markedly elevated PD, as subsequently shown in sweat ducts. However amiloride, a potent blocker of sodium channels, reduced PD in both non CF and CF subjects to similar absolute levels, suggesting that the basis for the raised PD in airways is an increased rate of sodium absorption. Further evidence for such an abnormality has come from direct measurements of 22 Na + fluxes across CF bronchi,40 double-barelled microelectrode studies on sodium activity in single CF airway cells 41 and recent single channel data suggesting an increase in the open probability of CF sodium channels. However, it is clear that the CF airway epithelial cells also possess the same abnormality as identified in the sweat duct, namely chloride impermeability. This has been shown in vivo with a lack of response of nasal epithelium to perfusion with a low chloride containing solu-

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tion (non CF cells respond to the favourable gradient thus created for chloride exit from the cell and thus nasal PD will increase; no response is observed in CF patients). Again, microelectrode studies show no response of single CF airways cells to stimulation of chloride secretion.42 Finally, studies of single airway chloride channels have demonstrated that in CF these channels fail to open in response to stimulation mediated through the cAMP/protein kinase A pathway.43 Whilst the exact type of chloride channel which is defective in CF is presently under debate, undoubtedly this cAMP-related chloride transport defect, which appears to be present in all affected epithelia in CF, is regarded as the hallmark of the basic defect in this disease. With regard to other second messenger pathways, chloride stimulation through the protein kinase C pathway is also defective, but direct elevation of intracellular calcium levels produces chloride secretion in CF tissues.44 Thus, in airways both the increase in sodium absorption and the reduction in chloride secretion will tend to reduce the water content of airway surface fluids. Although not proven, it is considered likely that these changes will lead to the abnormalities of mucus and bacterial colonisation. In summary, the basic defect in CF is likely to focus on the abnormal functioning of CFTR as a cAMP-regulatable chloride channel. In the airways, the principal site of morbidity and mortality, sodium transport is also abnormal and may predominate in the causation of such pathology. Although the inter-relationship of these two defects is presently unclear, therapy may be directly aimed at reversing either or both of these abnormalities. Therapy based on the CF ion transport abnormalities Increased sodium absorption The diuretic amiloride, which acts as a potent sodium channel blocker in a variety of epithelia, would appear to be a logical first choice to reduce absorption in CF airways. Pilot studies indicated that following oral administration amiloride levels in airway surface liquid (ASL) do not reach sufficiently high concentrations. Therefore, attention focussed on the use of topically nebulised amiloride with initial dosing studies in sheep,45 an animal having similar ion transport characteristics to man. Airway PD was reduced immediately after inhalation of 6 mM amiloride, but by 30 minutes the effect was not significantly different to that of the vehicle. This correlated with a rapid rate of drug removal from ASL (ti 10.5 min), in vitro studies suggesting this was effected through active transport Amiloride at this concentration, also significantly increased the volume of ASL immediately following

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nebulisation, although sodium, potassium and chloride electrolyte concentrations were unaltered in the fluid. Thus, sufficient concentrations of amiloride can be delivered to airways to inhibit sodium and water absorption. The first reported study in man46 described the effect of a single nebulised dose of amiloride (1 mM) on mucociliary clearance (MCC) in 14 CF patients. Clearance was measured by a gamma camera following inhalation of "Tc-labelled red cells with a median diameter of 6 u,m, in response to inhalation of either amiloride or placebo in a single-blind crossover design. MCC one hour after amiloride was significantly increased, with a maximum effect at 30 minutes. The effect was principally seen in the central, rather than the peripheral parts of the lung, which may relate to more central deposition in the presence of bronchial obstruction. Cough clearance (CC) was also significantly improved. No significant changes were seen in spirometry, nor did amiloride alter blood pressure. This study was later expanded to include 23 CF patients, 6 of whom completed a 3 week trial of twice daily amiloride following which acute measurements and CC were again repeated.47 MCC was significantly greater in the amiloride treated group after 3 weeks and tended to be faster than in the patients only exposed to a single inhalation. CC followed a similar pattern, except that following 3 weeks of amiloride there was a more marked (and significant) increase in clearance in comparison to that following a single inhalation. Interestingly, amiloride only significantly improved either MCC or CC in patients without a history of previous pneumonic illness (defined as an episode of fever accompanied by increased cough and sputum production in association with documentation of pneumonia on a chest radiograph). Also, a significant improvement in CC was only seen in younger patients (aged 14 or less), the authors suggesting that these factors emphasize the use of amiloride at the earliest possible stage in CF. Measurements of sputum viscoelasticity showed a significant decrease with amiloride whilst sputum sodium concentration was significantly increased, although sputum weight was not altered. Increased levels of bacteria were noted in the amiloride group, but further quantification was not provided. No side effects were noted in any of the patients. These initial studies clearly warranted further assessment of amiloride and a double-blind crossover trial of amiloride versus placebo has recently been reported.48 18 patients were originally recruited of whom 14 completed the study. The subjects were adults (mean age 25 years, range 18—37), with an average of approximately one hospital admission in the preceding year. All 14 patients were colonised with Pseudomonas aeruginosa and 8 with Staphylococcus aureus. Mean

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forced vital capacity (FVC) of the group was 81% of predicted and forced expiratory volume in one second (FEVj) 60% of predicted. Treatment periods were of 25 weeks duration and each was preceded by 10 to 14 days of parenteral tobramycin and ceftazidime. Thereafter, all respiratory treatment was withdrawn during the study period. A 2—4 week washout separated the two treatment periods. Suitable allowances were made for acute disease exacerbations. Measured indices of outcome included lung function, chest radiograph, sputum microbiology, routine blood and urine analysis as well as 24 hour urinary aldosterone excretion. Sputum samples were taken from 6 patients at the end of each period. Amiloride (5 mM) or placebo were nebulised four times daily by each patient. Spirometry declined in both amiloride and placebo groups. FEVi did not significantly differ, with a loss of 3.2 ml/day for placebo and 2.1 ml/day for amiloride. However, the decline in FVC was significantly greater in the placebo group (3.4 ml/day) compared to the amiloride group (1.4 ml/day). Indices of the mechanical impedance of mucus were significantly lower in the amiloride treated group, whilst calculated MCC and CC were significantly increased in these patients. Interestingly, rheological values of sputum following amiloride were similar to values for mucus previously described from uninfected human trachea and bronchus. No differences were found in bacterial densities of either group nor was there evidence of pulmonary or systemic toxicity. Similarly, the number of therapeutic interventions (oral antibiotics, bronchodilators and prednisolone) did not differ in either group. Whilst these are encouraging preliminary findings, two aspects require further comment. Firstly, prior to the start of the trial the 14 adult patients appeared to have relatively stable disease, requiring only, on average, one hospital admission in the previous year. A rate of decline of FVC of approximately 3.5 ml/day in the placebo group in the presence of a mean starting value of 3.71, would indicate a poorer than expected prognosis. However, the design of the trial, with patients having all other pulmonary therapy withheld, presumably contributed to these values. Secondly, whilst the likely target of amiloride is sodium and water movement, this agent has been shown to have numerous other properties. With respect to ion transport, amiloride can inhibit Na+/Ca2 exchange, Na+-glucose cotransport and the Na+/K+/ATPase, although the IC50 values for these are in the millimolar range.49 However, the Na+/H+ antiport is more sensitive to amiloride, as are T-type calcium channels,50 although to date neither have been described in human airway epithelial cells. Amiloride also possesses antimicrobial actions, being synergistic with tobramycin for Pseudomonas cepacia and bacteriocidal to haemolytic streptococci. It can also suppress the production of

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pyocyanin, one of the toxins produced by Pseudomonas aeruginosa.51 Furthermore, amiloride has anti-inflammatory properties52 and can inhibit DNA synthesis by intercalating into DNA and inhibiting DNA topoisomerase HI.53 Two other effects of amiloride are worthy of note. Firstly, it can inhibit urokinase54 in turn an activator of plasminogen, the precursor of the proteolytic enzyme plasmin. Urokinase is a product of alveolar macrophages and epithelial cells and decreased activity of this activator has been demonstrated in the adult respiratory distress syndrome. Urokinase is probably also important in the clearance of pulmonary fibrin, as well as of lung haemorrhage. Secondly, amiloride has been shown to antagonise the bacteriocidal effects of certain antistaphylococcal antibiotics, suggesting that amiloride should be not used during conventional treatment for Staphylococcus aureus related exacerbations.55 Clearly, larger studies are now warranted, both to try to confirm and extend these initial encouraging results, as well as to provide reassurance on the described safety aspects. Pilot trials of delivery of amiloride using either a jet or ultrasonic nebuliser have been completed.56 The ultrasonic nebuliser was able to deposit almost double the quantity of amiloride more rapidly (4—5 min for complete deposition) than the jet nebuliser (7-8 min) and was preferred by the patients. The jet nebuliser is being used in a double-blind crossover study (6 month for each of amiloride and placebo) with acute measurements of mucociliary clearance at the end of each of the study periods. This study is due for completion in April 1992. Reduced chloride secretion Chloride secretion probably provides a quantitatively less important contribution to airway ion transport than the absorption of sodium. Thus, attempts at reducing sodium absorption in CF should remain the first priority. However, a combination of a chloride secretagogue with amiloride would be likely to maximise ion transport related therapy for these patients. Furthermore, in the presence of amiloride, cellular dnving forces which allow for chloride transport, are altered in favour of increased secretion. As discussed in the previous section, chloride secretion mediated through the protein kinase A and C pathways is defective in CF. However, that produced by elevation of intracellular calcium appears to function normally. Several agents are known to act through this pathway, with bradykinin and the triphosphate nucleotides ATP and UTP being perhaps the most clinically relevant Using cultured airway epi-

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thelial cells, Mason et al 57 have shown that both nucleotides can produce an increase in chloride secretion (and intracellular calcium) in CF cells, although 30% of tested cells did not demonstrate such a response. Measurements in vivo using nasal PD as a marker of chloride secretion58 showed an increase in PD to both ATP and UTP in CF patients. Interestingly, the PD rose to similar levels in non CF and CF subjects suggesting the restitution of the previously absent chloride permeability in CF airways. The PD response was maintained during perfusion of the drugs into the nasal cavity, although data regarding the time course of action following cessation of topical drug application have not been published. Thus, limited applications of therapy directly related to the described ion transport defects have been attempted, with initially encouraging results. At present a combination of amiloride and a chloride secretagogue acting via elevation of intracellular calcium would appear to be theoretically the optimal treatment. ANTI-INFLAMMATORY APPROACHES Theory {see Warner, this issue) There is overwhelming evidence that the inflammatory processes directed against the colonising organisms in the lungs not only fail to eradicate the organisms but actually cause local tissue damage. The failure to eradicate the organisms is in part due to the fact that local conditions appear to favour colonisation and growth, in part due to various defenses employed by the bacteria, eg alginate production and microcolony formation, and in part due to the products of inflammation themselves. In particular the proteolytic enzymes released by inflammatory cells may break down some immune products and so actually render these immune processes ineffective. For example, neutrophil elastase59 cleaves chemotactic factors, breaks down some complement components, and inactivates immunoglobulins. In this way an inflammatory response can be considered as excessive in that it actually inhibits its own antimicrobial actions. At the same time many inflammatory mediators, once released may damage local host tissues and set up a cycle of chronic inflammatory damage and scarring. This damage then favours further bacterial growth as local mechanisms of bacterial inhibition and clearance are themselves impaired or destroyed. While many mediators are released during airway inflammation recent attention has focussed on neutrophil elastase and free radicals. A number of ways of countering this inflammatory damage have been proposed. These range from relatively specific therapy directed at

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proteolytic enzymes such as alpha-1-antitrypsin to more general antiinflammatory drugs both steroidal (eg prednisolone) and non-steroidal (eg ibuprofen). The evidence in favour of these three drugs are discussed below. The same logic can be extended to antioxidants such as gluthathione and to immune therapy with drugs such as methotrexate, azathioprine, or cyclosporin A. These latter agents have not yet been evaluated in CF. For all of these anti-inflamamtory and anti-immune approaches there are theoretical risks of uncontrolled infection. Prednisolone Corticosteroids are powerful anti-inflammatory drugs with some immunosuppressive properties. They can also affect any co-existing bronchospasm and so assist mucociliary clearance. Prednisolone has been frequently used as part of the treatment of CF but usually in low doses for short periods. This has been in part because of the risks of long term side effects and in part because of the possibility that it might worsen the respiratory infection. In 1985 Auerbach et al 60 reported a double blind trial in which children with CF and relatively mild lung disease received prednisolone 2 mg/kg on alternate days or placebo for 4 years. Prednisolone was associated with better lung function and weight, fewer hospital admissions and less evidence of immune activity as judged by lower IgG levels. Surprisingly this relatively high dose therapy was associated with few side effects, in particular, with no adverse effect on growth. This study led to considerable interest, as well as some scepticism about the apparent freedom from side effects, and a large multicentre double blind study was set up comparing placebo with 1 mg/kg and 2 mg/kg of prednisolone. The patients were monitored every 3 months and an interim analysis of results was performed every 6 months by an unblinded ombudsman. After nearly 3 years the interim analysis showed an unacceptable level of side effects (glucose intolerance, cataract, growth retardation) in the 2 mg/kg group and this part of the trial was abandoned.61 Until the final results are available chronic prednisolone treatment cannot be recommended. An alternative approach using chronic inhaled corticosteroids is also being evaluated but has not yet been reported. The inhaled route is likely to cause fewer side effects but has the theoretical disadvantage that the airways most affected by inflammation are likely to be the most difficult to reach.

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Ibuprofen This drug, in common with many other non-steroidal anti-inflammatory agents acts by inhibiting arachidonic acid metabolism and so interfering with the production of prostaglandins and leukotrienes at sites of inflammation. Preliminary studies in animals have shown some evidence of protection of the lungs from chronic inflammatory damage62 and clinical trials are now in progress. Alpha-1 -antitrypsin This is the most important of a number of antiproteases which protect the lungs from proteolytic enzymes. While its deficiency was originally associated with lung disease in the form of emphysema, it is now clear that low circulating levels of alpha-1-antitrypsin also predispose to bronchiectasis. Furthermore, in chronic pulmonary inflammation the amount of alpha-1-antitrypsin, although normal, appears to be inadequate to balance the amount of proteolytic enzyme released. Since replacement alpha-1-antitrypsin therapy may be valuable in the deficiency state considerable progress has already been made in investigating this form of therapy. Human alpha-1-antitrypsin can be prepared from donor blood and both infusions and aerosol delivery have been shown to provide normal levels of alpha-1-antitrypsin in the lung epithelial lining fluid (ELF) in alpha-1-antitrypsin deficient subjects. Furthermore, the alpha-1-antitrypsin gene has been cloned and bacterial insertion may eventually provide a pure source of human alpha-1-antitryspin. In CF preliminary studies McElvaney and colleagues63 have shown: (a) Normal levels of alpha-1-antitrypsin in ELF. (b) High levels of neutrophil elastase in ELF which swamp the alpha1-antitrypsin activity. (c) Aerosol alpha-1-antitrypsin elevates the levels in ELF 3 to 6 fold and the levels remain approximately twice normal 12 hours after administration. (d) Neutrophil killing of Pseudomonas is inhibited by ELF from CF patients (presumably because of the excess neutrophil elastase) but returns to normal following aerosol treatment with alpha-1antitrypsin. Both the theory as well as these preliminary studies suggest a possible role for alpha-1-antitrypsin in modifying the airway inflammation and enhancing bacterial killing in CF. There are not, however, as yet any supporting clinical data. Furthermore, Pseudomonas colonisation occurs in CF before there is gross inflammation and this treatment might only

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be appropriate for the more severely affected patients, in whom many other factors are contributing to their deterioration. An alternative anti-protease is secretory leukoprotease inhibitor (SLPI). This is probably a less important system than alpha-1-antitrypsin but is also subject to much current investigation. DNase Uninfected sputum consists of a complex mixture of glycoproteins, water and electrolytes. When sputum becomes infected a large number of additional substances derived both from bacteria and from the host's inflammatory response change its physical and chemical properties. In particular DNA, which is derived chiefly from neutrophils, is found in relatively high levels and makes the sputum more viscous and difficult to clear from the lungs. The gene from DNase I, the predominant enzyme responsible for the breakdown of DNA, has been cloned and recombinant human DNase (RhDNase) is now available. This enzyme has been studied for its possible benefit in liquifying CF sputum and convincingly increases the pourability and reduces the viscosity of sputum derived from chronically infected CF subjects in a time and dose dependent way.64 These preliminary data are encouraging and clinical studies have begun in the hope that it will be possible to improve sputum clearance at least during infectious exacerbations and so limit the duration of severity of the illness. Even more exciting would be the possibility of improving lung function by removing inspissated secretions or preventing the long term decline of lung function with chronic use. These optimistic possibilities must be set against the history of DNase therapy which is in no way new. Bovine pancreatic DNase has been available for 40 years and has been subjected to a number of trials. While benefits have been claimed for its use as an aerosol treatment for pneumonia and cystic fibrosis, the evidence to support any clinical benefit is slight.65- 66 A well designed trial of bovine DNase in purulent bronchitis showed improvement in sputum viscosity but no benefit in terms of lung function or other clinical parameters.67 Similarly, a trial in CF showed improved viscosity but clinical benefit was only reported in 4 of the 10 patients who took part. The author of this study claimed a major benefit of DNase especially in more seriously ill patients but the treatment never caught on and so cannot have been dramatically successful. The fact that the DNase was of bovine rather than human origin may not matter as the viscosity and pourability studies quoted above showed similar results with enzymes from both sources. Perhaps the most important problem with earlier trials of DNase was inadequate

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aerosol technology. In summary, DNase therapy is logical and certainly merits a second look. It will however, be important for the relatively inexpensive bovine DNase to be compared widi the very expensive recombinant human form. ETHICAL CONSIDERATIONS The more fundamental treatments such as gene and ion transport therapy will take many years to develop and their assessment will raise new ethical issues. They will, at least in theory, be most likely to benefit die patient if they are given very early, and ideally should be started before the lungs become colonised by bacteria and before vicious cycles of inflammatory damage begin. Indeed, it may well be that these treatments will prove relatively ineffective once the lungs are damaged since in this situation persistent bacterial growth is a consequence of this damage and its secondary effect on lung clearance mechanisms rather than due to the genetic defect itself. These arguments are particularly worrying as there is a natural preference to try out new treatments on those with the most severe disease who are most in need of new drugs and who may therefore be ready to accept the attendant risks. If it is accepted that pre-symptomatic patients are the most likely to benefit then difficult ediical issues must be raised. Will it be justified to give new treatments to children for many years in order to see whether lung disease is delayed or prevented? In the past when CF was almost always fatal in early life this approach would have been more easily accepted than today when 80% of patients with CF survive to the age of 20 and many are virtually free of symptoms. If the prognosis continues to improve then these issues will become even more difficult, particularly with entirely new treatments, such as gene therapy. In this situation a short term safety record would not be particularly reassuring and a long term safety record could only be obtained by long term studies in man. Nevertheless, in spite of the formidable difficulties, both scientific and ethical, the future has never looked brighter for people with CF. Not only are a number of entirely new treatments being very actively researched which may prevent lung damage, but progress in transplantation is giving new hope to those with end stage disease. While for those in between, conventional treatment is leading to an ever better outlook. ACKNOWLEDGEMENTS We would like to thank Steve Hart for helpful discussions and the Cystic Hbrosis Research Trust for its support over many years.

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