LEADING ART ICLE
Drugs 43 (4): 431-439. 1992 0012-666 7/92/0004-0431/$04.50/0 © Adis International Limited. All rights reserved. DRU1130
Drug Therapy in the 1990s
What Can We Expect for Cystic Fibrosis?
Richard C. Boucher Cystic Fibrosis/Pulmonary Research Center and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Cystic fibrosis research evolved rapidly in the 1980s. Great advances have been made, particularly in the elucidation of the molecular and cellular pathophysiology of the disease, although there were relatively fewer advances in its clinical therapy. This review provides a brief description of the molecular pathophysiology and the clinical ramifications of the disease. It focuses on the lung, because damage to this organ is the leading cause of morbidity and death in cystic fibrosis patients. The therapeutic strategies are reviewed in terms of new pharmacological approaches to the treatment of this disease, the potential use of protein replacement therapy, and finally the use of gene-transfer strategies to modify the disease.
1. Pathogenesis 0/ of Cystic Fibrosis The prevailing hypothesis is that abnormal ion transport is a direct consequence of the abnormal cystic fibrosis gene product and leads to abnormalities in the volume and composition of epithelial surface liquids. The identification of the cystic fibrosis gene was reported in 1989 (Kerem et al. 1989; Riordan et al. 1989; Rommens et a\. al. 1989). A 3-base pair deletion was identified as the mutation causing the majority of cystic fibrosis cases. From comparisons with known genes, the cystic fibrosis gene appears to code for a protein in the so-called' ATP binding cassette' (ABC) family (Hyde et a\. al. 1990). These
proteins exhibit a motif of 6 transmembrane spanning regions, followed by a nucleotide binding fold, followed by a repeat of this motif. Because many members of this class of protein are ATP-consuming solute pumps, it has been speculated that the protein product of the cystic fibrosis gene, known as the cystic fibrosis transmembrane regulator (CFTR) is functionally similar to these proteins (Hyde et al. 1990). However, recent data, employing the cloned gene for CFTR expressed in heterologous cell systems, have raised the possibility that CFTR may in fact be an anion (CI-) channel (Anderson et al. 1991; Kartner et al. 1991). The phenotypic expression of cystic fibrosis at the cellular level has been intensively explored over the past 10 years. In brief, it appears that cystic fibrosis is a disease which targets the epithelia that line affected organ systems, e.g. lung, pancreas, gastrointestinal tract and reproductive systems. A common feature of all affected epithelia is that cystic fibrosis cells appear to exhibit a relative impermeability to cellular Cl- ion flow. Most importantly, there is defective regulation of plasma membrane Cl- permeability by intracellular signal transduction systems operating through cyclic adenosine monophosphate (cAMP)-dependent kinase (PKA) and protein kinase C (PKC) [Boucher et a\. al. 1989; Li et al. 1988; Schoumacher et al. 1987]. Cl- permeability, however, can be activated in both cystic fibrosis and normal cells through signal systems that modulate intracellular Ca++ ([Ca++]i)
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levels (Boucher et al. 1989). It is not yet clear whether or not the Cl- channel that responds to elevations in [Ca++]j is the same channel that fails to respond to PKA and PKC in cystic fibrosis, or represents a different class of channels. In airway epithelia, the defective Cl- channel is located on the apical plasma membrane and limits the capacity to secrete Cl- ions towards the airway lumen. Other transport abnormalities have been identified in specific affected epithelia. For example, in airway epithelia the rate of absorption of Na+ ions from the airway luminal compartment to the blood compartment is raised 2- to 3-fold in cystic fibrosis, compared with normal airway epithelia (Boucher et al. 1986, 1988). The projected functional consequence of this abnormally raised rate in cystic fibrosis airways is a reduced volume of water in airway surface liquids. The functional consequence of this defect adds to the Cl- impermeability dysfunction.
2. Clinical Expression of Cystic Fibrosis The clinical expression of cystic fibrosis reflects the dysfunction of the affected epithelia. In the gastrointestinal tract, dehydrated interluminal intestinal contents lead to blockage of the intestine, socalled meconium ileus. In the pancreas, the inability of the pancreatic duct to secrete sodium bicarbonate and water results in failure to remove activated enzymes from the pancreatic acinar region, with consequent autodigestion of the pancreas. In the lung, the reduced wate( content of se-cretions alters the biorheological properties of airway secretions unfavourably, hindering their clearance from the lung and leading to infection. Indeed, infection of the lung accounts for more than 95% of deaths from cystic fibrosis (Wood et al. 1976). In this regard, a peculiar and unexplained feature ofthe disease is the predilection of the diseased lung for specific and characteristic bacteria flora, e.g. Staphylococcus aureus and Pseudomonas aeruginosa.
3. Pharmacological Approaches The goals for pharmacological therapy are 2-fold: (a) prevention of the development of abnormal airway secretions and lung infection, and (b) treatment of preexisting airways infection and inflammation. 3.1 Preventive Therapy The pancreatic insufficiency that results from the cystic fibrosis epithelial defect is typically treated successfully with oral replacement enzyme therapy. Intestinal obstruction, which occurs intermittently, is usually successfully treated with osmotic (radiocontrast) enemas. Over the first 6 months to several years of life, chronic and unremitting infections develop as a consequence of abnormalities in clearance and possibly of other properties of airway secretions. Thus, if the underlying ion transport defects can be successfully treated, it is feasible to expect that the development of the infectious and inflammatory components of cystic fibrosis lung disease can be prevented. New drugs targeted to 1 or both ion transport abnormalities detected in the diseased lung, the Na+ and the Cl- transport defects, would appear to be rational candidates for preventive therapy. 3.1.1 Sodium Transport Inhibitors Amiloride is a Na+ channel-blocker that has been used clinically as a K+-sparing diuretic. Because of its channel-blocking properties, tests were made on the activity of this compound in blocking transepithelial Na+ transport in human bronchi. A series of studies revealed that amiloride is an effective inhibitor of transepithelial Na+ transport in human airways in vivo and in vitro (Knowles et al. 1981, 1984). Formal dose-effect studies demonstrated a maximum effect at approximately 0.1 mmol/L, with an effective 50% inhibitory concentration (ECso) of approximately 1 ~mol/L. Amiloride is effective when exposed to the apical surface of the airway epithelial cell, the membrane containing the electrodiffusive Na+ channels, and
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is ineffective when administered to the basolateral (blood-facing) cell surface. A combination of in vivo and in vitro studies defined a profile of efficacy and potency in cystic fibrosis airway epithelia similar to those observed in healthy volunteers (Knowles et al. 1981; Waltner et al. 1987). The effectiveness of amiloride in blocking Na+ transport in human airway epithelia suggested that this compound could potentially be of therapeutic value in slowing the accelerated Na+ transport rates in cystic fibrosis airway epithelia. However, because amiloride is effective only from the luminal surface, therapeutic delivery of the drug to the airways surface requires aerosol administration. Because no diuretic of any class had ever been delivered to the lung by the aerosol route when these in vivo studies were initiated, this required investigation before the efficacy of amiloride in treating cystic fibrosis lung disease could be tested. Initially, it was important to establish that amiloride could be delivered in effective concentrations by the aerosol route (Waltner et al. 1987). This requirement led to the deve,opment of techniques to sample the small volumes « 1 /-LI) of liquids on the airways surface, in order to measure drug deposition and half-life. A technique employing filter paper pledgets housed in protective catheters that could be positioned on airway surfaces via a bronchoscope was developed for this purpose (Mentz et al. 1986). These studies established that effective concentrations of amiloride (0.1 mmoljL) could be delivered on to the airway surfaces via nebulisation of liquid solutions (3 mmoljL in aerosol canisters), and that the half-life of the compound in the airway surface liquid compartment, determined by radiotracer loss from the airway surface, was about 35 to 40 min. Similar data were obtained from both cystic fibrosis and normal patient populations (Waltner et al. 1987). On the basis of the drug deposition data, a series of safety studies was then performed to test whether aero soli sed amiloride deposited in concentrations that effectively inhibit Na+ transport was safe in healthy volunteers. It was found that inhaled amiloride had no short term effects on lung function, nor could adverse effects on pulmonary function
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or body fluid volume status be detected during 1 month of 4 times daily amiloride inhalation in healthy volunteers (Waltner et al. 1986). When these studies were complete, a doubleblind, crossover trial was performed in adult cystic fibrosis patients to study both the safety and the efficacy of amiloride administered 4 times daily for 6 months as an aerosol (Knowles et al. 1990). In cystic fibrosis patients with preexisting lung disease, it was found that the disease-induced loss of lung vital capacity [forced vital capacity (FYC)] was slowed by approximately 60% when patients were inhaling amiloride compared with vehicle (Knowles et al. 1990). Evaluation of the biorheological properties of sputum suggested that amiloride induced a normalisation of sputum viscoelastic properties, which would facilitate mucociliary clearance. These findings are congruent with the results of both short and long term (3 weeks) studies in cystic fibrosis patients as· young as 7 years, which showed that amiloride increased mucociliary clearance (App et al. 1990; King et al. 1990; Kohler et al. 1986). Amiloride appears to be the first compound that beneficially affects the course of established cystic fibrosis lung disease by targeting an abnormal electrolyte transport process characteristic of the cystic fibrosis airway epithelium. Studies are continuing with the aim of developing the appropriate pharmacokinetic information to test the hypothesis that amiloride delivered to patients before the onset of lung disease is protective. Such studies are in the design phase in both North America and Europe. 3.1.2 CI- Channel 'Openers' Given the prevalence ofCl- permeability defect in cystic fibrosis epithelia, mechanisms to restore the activity of this pathway are of obvious importance. Interestingly, acute activation of apical membrane Cl- permeability does not generate Clsecretion in cystic fibrosis or normal airway epithelia (Willumsen et al. 1989). To generate the electrochemical driving forces that produce Cl- secretion, human airway epithelia must be pretreated with amiloride. Thus, if the goal of activating Clchannels in airway epithelia is to induce Cl- secretion, combination therapy with amiloride may
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be required. It is possible, however, that long term activation of the Cl- permeability of the apical membrane may restore the balance in both Na+ and Cl- transport across airway epithelia. Two approaches, focused on different cellular regulatory mechanisms, are available for activating Cl- channels in cystic fibrosis airway epithelia. The first approach is based on the observation that elevations in [Ca++]j initiate Cl- secretion in human airway epithelia. The first clues that this was so were detected in studies of isolated Clchannels in patch clamp in vitro studies and subsequently in intact epithelial monolayers using Ca++ ionophores (Frizzell et al. 1986; Willumsen & Boucher 1989). Given the likelihood that Ca++ ionophores cannot be delivered safely to the lung by the aerosol route, a search has begun for other ways to raise [Ca++]j and initiate Cl- secretion. It has recently been observed that cell surface receptors linked to phospholipase C prom'ote inositol triphosphate (IP3) mediated intracellular Ca++ release and activation of plasma membrane Ca++ permeability, resulting in raised [Ca++]j levels and hence Cl- secretion in human airway epithelia. These actions have been observed in cystic fibrosis airway epithelia exposed to histamine or bradykinin (Boucher et al. 1989). Bradykinin may be the more interesting compound because receptors for this agonist are present on the luminal surface of airway epithelial cells. However, administering this compound to airways by aerosol may prove to be difficult because of irritant effects in the lung. In general, it would appear that classes of compounds that activate Cl- secretion via cell surface receptor [Ca++]j-gated mechanisms may be useful therapeutic agents for the treatment of the Clchannel defect in cystic fibrosis. Problems with regard to the duration of drug action [e.g. due to rapid desensitisation of cell surface receptors and/or rapid regulation of [Ca++]j to basal levels by cellular Ca++ adenosine triphosphatases, (ATPases)] and the issue of safety, specifically with respect to maintenance of raised [Ca++]j in epithelial cells, must be addressed before efficacy studies can be thought of. Other compounds may raise [Ca++]j and be effective Cl- secretagogues of cystic fibrosis tissue by
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a different mode of action. For example, duramycin is an antibiotic compound that appears to raise [Ca++]j and be an effective Cl- secretagogue in cystic fibrosis epithelia (Cloutier et al. 1988). Duramycin raises [Ca++]j by a non-IP3-dependent mechanism. Preliminary data indicate that this compound, like thapsigargin (Kwan et al. 1990), may inhibit intracellular organelle (calciosome) Ca++ ATPases with release of Ca++ from these stores into the cytoplasm. The safety and long term efficacy of this class of compounds has not yet been tested. The second mechanism for activating Cl- secretion in cystic fibrosis airway epithelia centres on recent observations that triphosphate nucleotides may directly regulate Cl- channel activity in human airway epithelia via direct ligand gating mechanisms similar to those reported for channels in neurons. In a series of studies, triphosphate nucleotides were shown to be effective Cl- secretagogues in cystic fibrosis airway epithelia (Mason et al. 1991). Although the assignment of the receptor mediating this response to a unique class is difficult because of the absence of selective antagonists, rank order potencies reveal that the pyrimidine uridine triphosphate (UTP) is as effective as adenosine triphosphate (ATP) in initiating Cl- secretion, suggesting that the receptor is not of the classical P2X or P2Y class (Burnstock & Warland 1987). Activation of these receptors does raise [Ca++]j by IP3-dependent mechanisms, indicating that this may be one potential mode of action of these compounds. However, a series of whole cell current clamp and excised patch clamp studies focusing on airway epithelial channels showed that the activation of Cl- channels by ATP is independent of Ca++ (Stutts et al. 1991). Further, these studies suggested that the Cl- channels activated by ATP biophysically resembled the class of channels activated by cAMP and PKC, and not those activated by Ca++ (Cliff & Frizzell 1990). The fact that ATP is more effective in cystic fibrosis than in normal airway epithelia in initiating Cl- secretion indicates that it acts directly, and bypasses the activation defect of the Cl- channel affected by cystic fibrosis. If this is the case, then ATP may directly
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restore the function of the defective protein in cystic fibrosis airway epithelia. Because of these findings, the activity of ATP and UTP was assessed in cystic fibrosis patients by local superfusion techniques in vivo (Knowles et al. 1991). In brief, it was found that both compounds were effective Cl- secretagogues in cystic fibrosis patients and in healthy volunteers, with both nucleotides being more effective in the former. The increased effectiveness reflected the fact that ATP or UTP restored the missing basal Cl- permeability in cystic fibrosis and activated the Cl- secretory rates to final levels that were not different in the 2 groups. The findings indicate that these compounds may be extremely useful in the development of pharmaceutical agents that could effectively activate the abnormal CFfR protein and, consequently, prevent the development oflung disease in patients with cystic fibrosis. As with amiloride, the use of this class of compounds via the aerosol route has not been explored extensively. It is known that inhaled ATP can be hydrolysed rapidly to adenosine, which is irritating to the lungs of asthmatics (Cushley et al. 1983). Because UTP is hydrolysed to uridine, which is not usually recognised by the adenosine receptor, pyrimidine base compounds may be preferable. An alternative choice would be the development of the poorly hydrolysed ATP analogues e.g. ATP 'Y5. A concern is that extracellular triphosphate nucleotides also stimulate granule discharge from airway luminal goblet cells (Davis et al. 1991), so that these compounds could actually increase mucus secretion. Thus, a goal for the development of nucleotides for the treatment of cystic fibrosis is to develop compounds that selectively target the receptor associated with the Cl- channel. 3.2 Treatment of Preexisting Disease Established cystic fibrosis lung disease is manifested by high concentrations of Pseudomonas and Staphylococcus bacterial species that inhabit the lumen of cystic fibrosis airways. Rarely, if ever, does infection spread beyond the airway lumen to the vascular compartment. Concomitant with infec-
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tion, an intense inflammatory response is focused on the airway. In part, this response is centred in the airway lumen, with large numbers of inflammatory cells and high concentrations of inflammatory mediators present. It is also manifested in the airway wall, with antigen-antibody complexes and other mediators leading to destruction of airway wall elements with the resultant development of bronchiectasis. Consequently, treatment of established cystic fibrosis disease focuses first, on eradicating the predominant bacteria species, and secondly, on reducing the inflammatory response which damages airway walls. 3.2.1 New Antibiotic Approaches The major target for new antibiotics is the Pseudomonas species. A typical regimen in acute exacerbations would include an aminoglycoside (e.g. tobramycin 7.5 mg/kg day) and a cephalosporin (e.g. ceftazidime 2g every 8 hours). Continued regular use of cephalosporin, semisynthetic penicillin and aminoglycoside antibiotics is regularly associated with new resistance patterns in Pseudomonas microorganisms. Certainly, new agents active against Pseudomonas species will be required in the 1990s; new compounds that could be administered by the oral route would be of particular importance. In a more general sense, a greater use of antibiotics able to be administered by aerosol will probably be seen. It is clear that this is a rational route for delivery because the nidus of infection in cystic fibrosis lungs is the· airway lumen. Thus, the development of vehicles that would delay drug release (and consequently sustain the duration of action in airway lumen) would appear to be warranted. 3.2.2 Anti-Inflammatory Agents Several anti-inflammatory drugs, including corticosteroids and ibuprofen, are currently being investigated for efficacy in reducing the incidence and severity of airway wall damage in cystic fibrosis. If these trials are promising, the 1990s could see some use of these agents to treat this manifestation of cystic fibrosis.
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More specific forms of anti-inflammatory activities also are becoming available for testing in the clinical arena. The large numbers of polymorphonuclear cells, or neutrophils, recruited into the airway lumen by persistent infection appear to be responsible for generating relatively high concentrations of free elastase activity in airway secretions. Consequently, aerosol delivery of recombinant human ai-antitrypsin and serum leucocyte proteinase inhibitor (SLPI) is being explored (McElvaney et al. 1991; Vogelmeier et al. 1991 ). Recently, it has been reasoned that the high concentration of DNA liberated by polymorphonuclear cells contributes to the abnormal viscoelastic properties of infected airway secretions. With the advent of recombinant human deoxyribonuclease (DNAse), the safety and efficacy of cleaving DNA molecules by this enzyme is being tested (Carswell et al. 1991). The approach appears rational, since the DNAse would remove the abnormal DNA contribution to sputum viscoelasticity but not perturb that of the native mucins that are required for effective transport. However, DNA may bind and inactivate high levels of cationic proteins (e.g. cathepsin G) that when liberated by DNAse might accelerate the damage of airway walls. Studies focusing on both short and long term safety of DNAse are therefore likely to be important.
4. Protein Replacement Strategies By analogy with haemophilia and genetic lung disease (e.g. ai-antitrypsin deficiency-induced emphysema) replacement of the defective cystic fibrosis protein with the normal product of the cystic fibrosis gene would appear to be a rational form of therapy. The key to the feasibility of such therapy awaits in part the results of studies designed to characterise the metabolism and location of the cystic fibrosis gene product. However, on the basis of the hypothesis that CFfR is localised in the lumen-facing (apical) plasma membrane, the site of the defective Cl- channel in airways epithelia, the practical issues with regard to protein replacement
can be considered. First, since CFfR is probably a large membrane-integral protein, expression of quantities of CFfR by recombinant technology suitable for pharmaceutical use will be challenging. Similarly, the purification of biologically active protein may also be difficult. Secondly, efficient delivery of the CFfR proteins to the lung by the aerosol route will require development. This route has delivered recombinant protein such as ai-antitrypsin and SLPI in active form to the lung (McElvaney et al. 1991; Vogel meier et al. 1991), but its use to deliver large hydrophobic proteins has not been reported. Presumably, some form of liposome-based delivery system will be required. Thirdly, the issue of safety must be addressed. If CFfR is a Cl- channel, then excessive protein delivery raises the issue of whether a cell can tolerate too many Cl- channels. Precise dose-effect relationships for efficacy and safety will be required before testing in humans.
5. Gene Therapy The concept of 'gene therapy' is potentially applicable to the treatment of cystic fibrosis lung disease. The basic premise, based on the observation that heterozygotes (carriers) are normal with regard to lung function, is that the introduction of the complementary DNA (cDNA) coding for the normal CFfR protein will convert affected cells to the heterozygous forms that function normally. Unlike the therapeutic approaches envisioned for the treatment of bone marrow disorders, it appears to be improbable that cells can be harvested from the cystic fibrosis lung, genetically corrected, and returned to the host. The complex branching pattern of the lung would appear to preclude this approach. However, it is possible that DNA vectors can be delivered for correction directly to the lung by aerosol or direct installation. Two general approaches to correction can be envisioned: the so-called transient, or nonintegrative, expression of the CFfR cDNA; and integrative approaches. Each approach has its own virtues and is discussed separately.
Cystic Fibrosis Therapy in the 1990s
5.1 Nonintegrative Approaches In transient expression approaches, the cDNA coding for CFTR will be introduced into affected cells in vectors that do not require chromosomal integration for transcription. Typically, expression of the foreign protein by nonintegrative approaches will last from days to weeks. One approach envisioned is to utilise a cDNA cloned into a plasmid vector encapsulated in a Iiposome that can be delivered to the airway cell via inhalation. Liposomes can carry a variety of plasmid vectors, including vectors that can promote nuclear targeting of foreign DNA. Liposomal technologies for the efficient transfer of genes have improved rapidly, but at present they probably do not approach the degree of efficacy required to reach a significant fraction of airway epithelial cells, although it must be noted that the proportion of the epithelial cell population that must be corrected is unknown. It is also not yet known whether there will be a requirement for selective targeting of a liposome to one epithelial cell type or another. However, it is clear that cells facing the airway lumen will require correction. Nonintegrative therapy can also be delivered to the airway epithelial cells by a variety of viral vectors. Adenoviral vectors (Berkner 1988) are candidates for the treatment of cystic fibrosis lung disease becau,se of their tropism for airway epithelial cells (Castleman 1985). Recombinant adenoviruses with viral genes deleted and replaced with CFTR cDNAs are feasible. Similarly, it is possible that vectors that are continually maintained episomally [e.g Epstein-Barr virus vectors (Yates et al. 1985)] can be considered. Finally, the vaccinia viral system has been utilised to express CFTR transiently in airway epithelia cells and correct the Cl- channel defect (Rich et al. 1990); however, the fact that these viruses are ultimately lytic make them unlikely candidates for gene therapy purposes. 5.2 Integrative Approaches The alternative strategy in gene therapy is to utilise vectors that promote integration of the foreign cDNA into the host genome. The obvious ad-
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vantage of this approach is that the corrective gene can be maintained indefinitely in progenitor cells and can ultimately be viewed as effecting a 'cure' of cystic fibrosis airways disease. The efficient vector presently available for this purpose is the retrovirus. Retroviruses deliver RNA into the host cell that is reverse transcribed into DNA and, via retroviral integrase activities, incorporate the foreign DNA into the host genome. Recent studies have indicated that retrovirally delivered normal CFTR cDNAs correct the cystic fibrosis apical membrane Cl- channel in polarised airway epithelial cells (Olsen et al. 1991). This result, plus the observation that correction can be maintained in progenitor cells which can differentiate into transporting epithelial cells in vitro for up to 6 months, indicates that this may be an efficient means of treating cystic fibrosis lung disease. Nevertheless, major hurdles remain before this form of therapy can be considered for the treatment of lung disease. The first problem is efficiency. Retroviruses infect only proliferating cells. A low efficiency of retroviruses in gene transfer in the lung can be anticipated, because fewer than 1% of all airway cells are replication competent at any one time. The simplest strategy to offset this problem would be repetitive exposure to virus. Methods of boosting the size of the replication competent pool, perhaps in response to mild airway injuries, may be useful adjuncts to increase efficiency. A second major problem is safety. Retrovirally introduced genes integrate into the host genome at random. Positional factors may affect the rate of transcription which, if high, could have direct toxic effects on cell viability, e.g. via too many plasma membrane channels. Alternatively, random integration may interrupt important relationships between growth/antigrowth genes and potentially lead to a risk of carcinogenesis. Both problem areas need intensive investigation before this form of therapy is suitable for human trials.
6. Conclusions It appears that a variety of novel therapeutic agents will become available for the treatment of cystic fibrosis lung disease in this decade. It is clear
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that currently available anti-inflammatory drugs, and newly available agents that affect the composition of mucus, can be tested for efficacy in ameliorating preexisting lung disease. A new generation of agents that target the ion transport defects of cystic fibrosia airway epithelia will also become available. These agents offer the exciting possibility of not only arresting the progression of cystic fibrosis lung disease in patients with existing disease, but preventing the onset of disease in children treated early. It is conceivable that both agents which block excessive Na+ absorption (e.g. amiloride) and new agents which may directly correct the Cl- channel defect (triphosphate nucleotides) may be extremely efficient and potentially safe in this regard. Indeed, these agents alone may be able to perturb cystic fibrosis lung disease sufficiently to effect a functional ·cure'. Finally, it is possible that genetic techniques for attacking this disease will become important. Expression and delivery of normal recombinant CFTR protein may be feasible; alternatively, DNA therapy may prove practical. It has already been shown that expression of the normal CFTR cDNA will correct a phenotypic defect of cystic fibrosis in complex airway epithelial preparations in vitro. Access to the target tissue by the aerosol route makes delivery of the gene therapy vectors to the lung appealing. It is likely that transfer efficiency and safety will determine whether or not this form of therapy is brought to the cystic fibrosis arena in the next decade.
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Effects of methacholine, thapsigargin and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. American Journal of Physiology 258: CIOO6-CI015, 1990 Li M, McCann JD, Liedke CM, Nairn AC, Greengard P, et al. Cyclic AMP - dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331: 358-360, 1988 Mason SJ, Paradiso AM, Boucher RC. Regulation of transepithelial ion transport and intracellular calcium by extracellular adenosine triphosphate in human normal and cystic fibrosis airway epithelium. British Journal of Pharmacology 103: 16491656, 1991 McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, et al. Aerosol "'I-antitrypsin treatment for cystic fibrosis. Lancet 337: 392-394, 1991 Mentz WM, Brown JB, Friedman M, Stutts MJ, Gatzy JT, et al. Deposition, clearance, and effects of aerosolized amiloride in sheep airways. American Review of Respiratory Disease 134: 938-943, 1986 Olsen JC, Johnson LG, Sarkadi B, Stutts MJ, Yankaskas JR, Boucher RC. Long term correction of chloride permeability defect by retrovirus transfer of CFTR cDNA to human airway epithelial cells. Abstract. Pediatric Pulmonology (Suppl. 6): p. 247, 1991 Rich DP, Anderson MP, Gregory RJ, Cheng SH, Paul S, et al. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347: 358-363, 1990 Riordan JR, Rommens JM, Kerem B-T, Alon N, Rozmahel R, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 10661073, 1989 Rommens JM, Iannuzzi MC, Kerem B-T, Drumm ML, Melmer G, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065, 1989 Schoumacher RA, Shoemaker RL, Halm DR, Tallant EA, Wal-
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lace RW, et al. Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature 330: 752-754, 1987 Stutts MJ, Chinet THC, Mason SJ, Fulton JM, Clarke LL, Boucher RC. Regulation of chloride channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP. Proceedings of the National Academy of Sciences, in press, 1991 Vogelmeier C, Hubbard RC, Fells GA, Schnebli H-P, Thompson RC, et al. Anti-neutrophil elastase defence of the normal human respiratory epithelial surface provided by the secretory leikoprotease inhibitor. Journal of Clinical Investigation 87: 482-488, 1991 Waltner WE, Boucher RC, Gatzy JT, Knowles MR. Pharmacotherapy of airway disease in cystic fibrosis. Trends in Pharmacological Sciences 8: 316-320, 1987 Waltner WE, Church NL, Gatzy JT, Boucher RC, Knowles MR. Toxicity and pharmacokinetics of acute amiloride aerosol in normal and cystic fibrosis subjects. 27th Annual Meeting of the Cystic Fibrosis Club. Abstracts 27: 121, 1986 Willumsen NJ, Boucher RC. Activation of an apical CI- conductance by ea 2+ ionophores in cystic fibrosis airway epithelia. American Journal of Physiology 256: C226-C233, 1989 Willumsen NJ, Davis CW, Boucher RC. Intracellular CI- activity and cellular pathways in cultured human airway epithelium. American Journal of Physiology 256: CI033-CI044, 1989 Wood RE, Boat TF, Doershuk CF. Cystic fibrosis. American Review of Respiratory Disease 113: 833-878, 1976 Yates JL, Warren N, Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313: 812-815, 1985
Correspondence and reprints: Prof. Richard C. Boucher Jr, Pulmonary Division, Department of Medicine, CB# 7020, 724 Burnett-Womack Building, University of North Carolina at Chapel Hill, Chapel Hill, Chapel Hill, NC 27599-7020, USA.
Drugs 43 (4): 431-439. 1992 0012-666 7/92/0004-0431/$04.50/0 © Adis International Limited. All rights reserved. DRU1130
Drug Therapy in the 1990s
What Can We Expect for Cystic Fibrosis?
Richard C. Boucher Cystic Fibrosis/Pulmonary Research Center and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Cystic fibrosis research evolved rapidly in the 1980s. Great advances have been made, particularly in the elucidation of the molecular and cellular pathophysiology of the disease, although there were relatively fewer advances in its clinical therapy. This review provides a brief description of the molecular pathophysiology and the clinical ramifications of the disease. It focuses on the lung, because damage to this organ is the leading cause of morbidity and death in cystic fibrosis patients. The therapeutic strategies are reviewed in terms of new pharmacological approaches to the treatment of this disease, the potential use of protein replacement therapy, and finally the use of gene-transfer strategies to modify the disease.
1. Pathogenesis 0/ of Cystic Fibrosis The prevailing hypothesis is that abnormal ion transport is a direct consequence of the abnormal cystic fibrosis gene product and leads to abnormalities in the volume and composition of epithelial surface liquids. The identification of the cystic fibrosis gene was reported in 1989 (Kerem et al. 1989; Riordan et al. 1989; Rommens et a\. al. 1989). A 3-base pair deletion was identified as the mutation causing the majority of cystic fibrosis cases. From comparisons with known genes, the cystic fibrosis gene appears to code for a protein in the so-called' ATP binding cassette' (ABC) family (Hyde et a\. al. 1990). These
proteins exhibit a motif of 6 transmembrane spanning regions, followed by a nucleotide binding fold, followed by a repeat of this motif. Because many members of this class of protein are ATP-consuming solute pumps, it has been speculated that the protein product of the cystic fibrosis gene, known as the cystic fibrosis transmembrane regulator (CFTR) is functionally similar to these proteins (Hyde et al. 1990). However, recent data, employing the cloned gene for CFTR expressed in heterologous cell systems, have raised the possibility that CFTR may in fact be an anion (CI-) channel (Anderson et al. 1991; Kartner et al. 1991). The phenotypic expression of cystic fibrosis at the cellular level has been intensively explored over the past 10 years. In brief, it appears that cystic fibrosis is a disease which targets the epithelia that line affected organ systems, e.g. lung, pancreas, gastrointestinal tract and reproductive systems. A common feature of all affected epithelia is that cystic fibrosis cells appear to exhibit a relative impermeability to cellular Cl- ion flow. Most importantly, there is defective regulation of plasma membrane Cl- permeability by intracellular signal transduction systems operating through cyclic adenosine monophosphate (cAMP)-dependent kinase (PKA) and protein kinase C (PKC) [Boucher et a\. al. 1989; Li et al. 1988; Schoumacher et al. 1987]. Cl- permeability, however, can be activated in both cystic fibrosis and normal cells through signal systems that modulate intracellular Ca++ ([Ca++]i)
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levels (Boucher et al. 1989). It is not yet clear whether or not the Cl- channel that responds to elevations in [Ca++]j is the same channel that fails to respond to PKA and PKC in cystic fibrosis, or represents a different class of channels. In airway epithelia, the defective Cl- channel is located on the apical plasma membrane and limits the capacity to secrete Cl- ions towards the airway lumen. Other transport abnormalities have been identified in specific affected epithelia. For example, in airway epithelia the rate of absorption of Na+ ions from the airway luminal compartment to the blood compartment is raised 2- to 3-fold in cystic fibrosis, compared with normal airway epithelia (Boucher et al. 1986, 1988). The projected functional consequence of this abnormally raised rate in cystic fibrosis airways is a reduced volume of water in airway surface liquids. The functional consequence of this defect adds to the Cl- impermeability dysfunction.
2. Clinical Expression of Cystic Fibrosis The clinical expression of cystic fibrosis reflects the dysfunction of the affected epithelia. In the gastrointestinal tract, dehydrated interluminal intestinal contents lead to blockage of the intestine, socalled meconium ileus. In the pancreas, the inability of the pancreatic duct to secrete sodium bicarbonate and water results in failure to remove activated enzymes from the pancreatic acinar region, with consequent autodigestion of the pancreas. In the lung, the reduced wate( content of se-cretions alters the biorheological properties of airway secretions unfavourably, hindering their clearance from the lung and leading to infection. Indeed, infection of the lung accounts for more than 95% of deaths from cystic fibrosis (Wood et al. 1976). In this regard, a peculiar and unexplained feature ofthe disease is the predilection of the diseased lung for specific and characteristic bacteria flora, e.g. Staphylococcus aureus and Pseudomonas aeruginosa.
3. Pharmacological Approaches The goals for pharmacological therapy are 2-fold: (a) prevention of the development of abnormal airway secretions and lung infection, and (b) treatment of preexisting airways infection and inflammation. 3.1 Preventive Therapy The pancreatic insufficiency that results from the cystic fibrosis epithelial defect is typically treated successfully with oral replacement enzyme therapy. Intestinal obstruction, which occurs intermittently, is usually successfully treated with osmotic (radiocontrast) enemas. Over the first 6 months to several years of life, chronic and unremitting infections develop as a consequence of abnormalities in clearance and possibly of other properties of airway secretions. Thus, if the underlying ion transport defects can be successfully treated, it is feasible to expect that the development of the infectious and inflammatory components of cystic fibrosis lung disease can be prevented. New drugs targeted to 1 or both ion transport abnormalities detected in the diseased lung, the Na+ and the Cl- transport defects, would appear to be rational candidates for preventive therapy. 3.1.1 Sodium Transport Inhibitors Amiloride is a Na+ channel-blocker that has been used clinically as a K+-sparing diuretic. Because of its channel-blocking properties, tests were made on the activity of this compound in blocking transepithelial Na+ transport in human bronchi. A series of studies revealed that amiloride is an effective inhibitor of transepithelial Na+ transport in human airways in vivo and in vitro (Knowles et al. 1981, 1984). Formal dose-effect studies demonstrated a maximum effect at approximately 0.1 mmol/L, with an effective 50% inhibitory concentration (ECso) of approximately 1 ~mol/L. Amiloride is effective when exposed to the apical surface of the airway epithelial cell, the membrane containing the electrodiffusive Na+ channels, and
Cystic Fibrosis Therapy in the 1990s
is ineffective when administered to the basolateral (blood-facing) cell surface. A combination of in vivo and in vitro studies defined a profile of efficacy and potency in cystic fibrosis airway epithelia similar to those observed in healthy volunteers (Knowles et al. 1981; Waltner et al. 1987). The effectiveness of amiloride in blocking Na+ transport in human airway epithelia suggested that this compound could potentially be of therapeutic value in slowing the accelerated Na+ transport rates in cystic fibrosis airway epithelia. However, because amiloride is effective only from the luminal surface, therapeutic delivery of the drug to the airways surface requires aerosol administration. Because no diuretic of any class had ever been delivered to the lung by the aerosol route when these in vivo studies were initiated, this required investigation before the efficacy of amiloride in treating cystic fibrosis lung disease could be tested. Initially, it was important to establish that amiloride could be delivered in effective concentrations by the aerosol route (Waltner et al. 1987). This requirement led to the deve,opment of techniques to sample the small volumes « 1 /-LI) of liquids on the airways surface, in order to measure drug deposition and half-life. A technique employing filter paper pledgets housed in protective catheters that could be positioned on airway surfaces via a bronchoscope was developed for this purpose (Mentz et al. 1986). These studies established that effective concentrations of amiloride (0.1 mmoljL) could be delivered on to the airway surfaces via nebulisation of liquid solutions (3 mmoljL in aerosol canisters), and that the half-life of the compound in the airway surface liquid compartment, determined by radiotracer loss from the airway surface, was about 35 to 40 min. Similar data were obtained from both cystic fibrosis and normal patient populations (Waltner et al. 1987). On the basis of the drug deposition data, a series of safety studies was then performed to test whether aero soli sed amiloride deposited in concentrations that effectively inhibit Na+ transport was safe in healthy volunteers. It was found that inhaled amiloride had no short term effects on lung function, nor could adverse effects on pulmonary function
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or body fluid volume status be detected during 1 month of 4 times daily amiloride inhalation in healthy volunteers (Waltner et al. 1986). When these studies were complete, a doubleblind, crossover trial was performed in adult cystic fibrosis patients to study both the safety and the efficacy of amiloride administered 4 times daily for 6 months as an aerosol (Knowles et al. 1990). In cystic fibrosis patients with preexisting lung disease, it was found that the disease-induced loss of lung vital capacity [forced vital capacity (FYC)] was slowed by approximately 60% when patients were inhaling amiloride compared with vehicle (Knowles et al. 1990). Evaluation of the biorheological properties of sputum suggested that amiloride induced a normalisation of sputum viscoelastic properties, which would facilitate mucociliary clearance. These findings are congruent with the results of both short and long term (3 weeks) studies in cystic fibrosis patients as· young as 7 years, which showed that amiloride increased mucociliary clearance (App et al. 1990; King et al. 1990; Kohler et al. 1986). Amiloride appears to be the first compound that beneficially affects the course of established cystic fibrosis lung disease by targeting an abnormal electrolyte transport process characteristic of the cystic fibrosis airway epithelium. Studies are continuing with the aim of developing the appropriate pharmacokinetic information to test the hypothesis that amiloride delivered to patients before the onset of lung disease is protective. Such studies are in the design phase in both North America and Europe. 3.1.2 CI- Channel 'Openers' Given the prevalence ofCl- permeability defect in cystic fibrosis epithelia, mechanisms to restore the activity of this pathway are of obvious importance. Interestingly, acute activation of apical membrane Cl- permeability does not generate Clsecretion in cystic fibrosis or normal airway epithelia (Willumsen et al. 1989). To generate the electrochemical driving forces that produce Cl- secretion, human airway epithelia must be pretreated with amiloride. Thus, if the goal of activating Clchannels in airway epithelia is to induce Cl- secretion, combination therapy with amiloride may
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be required. It is possible, however, that long term activation of the Cl- permeability of the apical membrane may restore the balance in both Na+ and Cl- transport across airway epithelia. Two approaches, focused on different cellular regulatory mechanisms, are available for activating Cl- channels in cystic fibrosis airway epithelia. The first approach is based on the observation that elevations in [Ca++]j initiate Cl- secretion in human airway epithelia. The first clues that this was so were detected in studies of isolated Clchannels in patch clamp in vitro studies and subsequently in intact epithelial monolayers using Ca++ ionophores (Frizzell et al. 1986; Willumsen & Boucher 1989). Given the likelihood that Ca++ ionophores cannot be delivered safely to the lung by the aerosol route, a search has begun for other ways to raise [Ca++]j and initiate Cl- secretion. It has recently been observed that cell surface receptors linked to phospholipase C prom'ote inositol triphosphate (IP3) mediated intracellular Ca++ release and activation of plasma membrane Ca++ permeability, resulting in raised [Ca++]j levels and hence Cl- secretion in human airway epithelia. These actions have been observed in cystic fibrosis airway epithelia exposed to histamine or bradykinin (Boucher et al. 1989). Bradykinin may be the more interesting compound because receptors for this agonist are present on the luminal surface of airway epithelial cells. However, administering this compound to airways by aerosol may prove to be difficult because of irritant effects in the lung. In general, it would appear that classes of compounds that activate Cl- secretion via cell surface receptor [Ca++]j-gated mechanisms may be useful therapeutic agents for the treatment of the Clchannel defect in cystic fibrosis. Problems with regard to the duration of drug action [e.g. due to rapid desensitisation of cell surface receptors and/or rapid regulation of [Ca++]j to basal levels by cellular Ca++ adenosine triphosphatases, (ATPases)] and the issue of safety, specifically with respect to maintenance of raised [Ca++]j in epithelial cells, must be addressed before efficacy studies can be thought of. Other compounds may raise [Ca++]j and be effective Cl- secretagogues of cystic fibrosis tissue by
Drugs 43 (4) 1992
a different mode of action. For example, duramycin is an antibiotic compound that appears to raise [Ca++]j and be an effective Cl- secretagogue in cystic fibrosis epithelia (Cloutier et al. 1988). Duramycin raises [Ca++]j by a non-IP3-dependent mechanism. Preliminary data indicate that this compound, like thapsigargin (Kwan et al. 1990), may inhibit intracellular organelle (calciosome) Ca++ ATPases with release of Ca++ from these stores into the cytoplasm. The safety and long term efficacy of this class of compounds has not yet been tested. The second mechanism for activating Cl- secretion in cystic fibrosis airway epithelia centres on recent observations that triphosphate nucleotides may directly regulate Cl- channel activity in human airway epithelia via direct ligand gating mechanisms similar to those reported for channels in neurons. In a series of studies, triphosphate nucleotides were shown to be effective Cl- secretagogues in cystic fibrosis airway epithelia (Mason et al. 1991). Although the assignment of the receptor mediating this response to a unique class is difficult because of the absence of selective antagonists, rank order potencies reveal that the pyrimidine uridine triphosphate (UTP) is as effective as adenosine triphosphate (ATP) in initiating Cl- secretion, suggesting that the receptor is not of the classical P2X or P2Y class (Burnstock & Warland 1987). Activation of these receptors does raise [Ca++]j by IP3-dependent mechanisms, indicating that this may be one potential mode of action of these compounds. However, a series of whole cell current clamp and excised patch clamp studies focusing on airway epithelial channels showed that the activation of Cl- channels by ATP is independent of Ca++ (Stutts et al. 1991). Further, these studies suggested that the Cl- channels activated by ATP biophysically resembled the class of channels activated by cAMP and PKC, and not those activated by Ca++ (Cliff & Frizzell 1990). The fact that ATP is more effective in cystic fibrosis than in normal airway epithelia in initiating Cl- secretion indicates that it acts directly, and bypasses the activation defect of the Cl- channel affected by cystic fibrosis. If this is the case, then ATP may directly
Cystic Fibrosis Therapy in the 1990s
restore the function of the defective protein in cystic fibrosis airway epithelia. Because of these findings, the activity of ATP and UTP was assessed in cystic fibrosis patients by local superfusion techniques in vivo (Knowles et al. 1991). In brief, it was found that both compounds were effective Cl- secretagogues in cystic fibrosis patients and in healthy volunteers, with both nucleotides being more effective in the former. The increased effectiveness reflected the fact that ATP or UTP restored the missing basal Cl- permeability in cystic fibrosis and activated the Cl- secretory rates to final levels that were not different in the 2 groups. The findings indicate that these compounds may be extremely useful in the development of pharmaceutical agents that could effectively activate the abnormal CFfR protein and, consequently, prevent the development oflung disease in patients with cystic fibrosis. As with amiloride, the use of this class of compounds via the aerosol route has not been explored extensively. It is known that inhaled ATP can be hydrolysed rapidly to adenosine, which is irritating to the lungs of asthmatics (Cushley et al. 1983). Because UTP is hydrolysed to uridine, which is not usually recognised by the adenosine receptor, pyrimidine base compounds may be preferable. An alternative choice would be the development of the poorly hydrolysed ATP analogues e.g. ATP 'Y5. A concern is that extracellular triphosphate nucleotides also stimulate granule discharge from airway luminal goblet cells (Davis et al. 1991), so that these compounds could actually increase mucus secretion. Thus, a goal for the development of nucleotides for the treatment of cystic fibrosis is to develop compounds that selectively target the receptor associated with the Cl- channel. 3.2 Treatment of Preexisting Disease Established cystic fibrosis lung disease is manifested by high concentrations of Pseudomonas and Staphylococcus bacterial species that inhabit the lumen of cystic fibrosis airways. Rarely, if ever, does infection spread beyond the airway lumen to the vascular compartment. Concomitant with infec-
435
tion, an intense inflammatory response is focused on the airway. In part, this response is centred in the airway lumen, with large numbers of inflammatory cells and high concentrations of inflammatory mediators present. It is also manifested in the airway wall, with antigen-antibody complexes and other mediators leading to destruction of airway wall elements with the resultant development of bronchiectasis. Consequently, treatment of established cystic fibrosis disease focuses first, on eradicating the predominant bacteria species, and secondly, on reducing the inflammatory response which damages airway walls. 3.2.1 New Antibiotic Approaches The major target for new antibiotics is the Pseudomonas species. A typical regimen in acute exacerbations would include an aminoglycoside (e.g. tobramycin 7.5 mg/kg day) and a cephalosporin (e.g. ceftazidime 2g every 8 hours). Continued regular use of cephalosporin, semisynthetic penicillin and aminoglycoside antibiotics is regularly associated with new resistance patterns in Pseudomonas microorganisms. Certainly, new agents active against Pseudomonas species will be required in the 1990s; new compounds that could be administered by the oral route would be of particular importance. In a more general sense, a greater use of antibiotics able to be administered by aerosol will probably be seen. It is clear that this is a rational route for delivery because the nidus of infection in cystic fibrosis lungs is the· airway lumen. Thus, the development of vehicles that would delay drug release (and consequently sustain the duration of action in airway lumen) would appear to be warranted. 3.2.2 Anti-Inflammatory Agents Several anti-inflammatory drugs, including corticosteroids and ibuprofen, are currently being investigated for efficacy in reducing the incidence and severity of airway wall damage in cystic fibrosis. If these trials are promising, the 1990s could see some use of these agents to treat this manifestation of cystic fibrosis.
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More specific forms of anti-inflammatory activities also are becoming available for testing in the clinical arena. The large numbers of polymorphonuclear cells, or neutrophils, recruited into the airway lumen by persistent infection appear to be responsible for generating relatively high concentrations of free elastase activity in airway secretions. Consequently, aerosol delivery of recombinant human ai-antitrypsin and serum leucocyte proteinase inhibitor (SLPI) is being explored (McElvaney et al. 1991; Vogelmeier et al. 1991 ). Recently, it has been reasoned that the high concentration of DNA liberated by polymorphonuclear cells contributes to the abnormal viscoelastic properties of infected airway secretions. With the advent of recombinant human deoxyribonuclease (DNAse), the safety and efficacy of cleaving DNA molecules by this enzyme is being tested (Carswell et al. 1991). The approach appears rational, since the DNAse would remove the abnormal DNA contribution to sputum viscoelasticity but not perturb that of the native mucins that are required for effective transport. However, DNA may bind and inactivate high levels of cationic proteins (e.g. cathepsin G) that when liberated by DNAse might accelerate the damage of airway walls. Studies focusing on both short and long term safety of DNAse are therefore likely to be important.
4. Protein Replacement Strategies By analogy with haemophilia and genetic lung disease (e.g. ai-antitrypsin deficiency-induced emphysema) replacement of the defective cystic fibrosis protein with the normal product of the cystic fibrosis gene would appear to be a rational form of therapy. The key to the feasibility of such therapy awaits in part the results of studies designed to characterise the metabolism and location of the cystic fibrosis gene product. However, on the basis of the hypothesis that CFfR is localised in the lumen-facing (apical) plasma membrane, the site of the defective Cl- channel in airways epithelia, the practical issues with regard to protein replacement
can be considered. First, since CFfR is probably a large membrane-integral protein, expression of quantities of CFfR by recombinant technology suitable for pharmaceutical use will be challenging. Similarly, the purification of biologically active protein may also be difficult. Secondly, efficient delivery of the CFfR proteins to the lung by the aerosol route will require development. This route has delivered recombinant protein such as ai-antitrypsin and SLPI in active form to the lung (McElvaney et al. 1991; Vogel meier et al. 1991), but its use to deliver large hydrophobic proteins has not been reported. Presumably, some form of liposome-based delivery system will be required. Thirdly, the issue of safety must be addressed. If CFfR is a Cl- channel, then excessive protein delivery raises the issue of whether a cell can tolerate too many Cl- channels. Precise dose-effect relationships for efficacy and safety will be required before testing in humans.
5. Gene Therapy The concept of 'gene therapy' is potentially applicable to the treatment of cystic fibrosis lung disease. The basic premise, based on the observation that heterozygotes (carriers) are normal with regard to lung function, is that the introduction of the complementary DNA (cDNA) coding for the normal CFfR protein will convert affected cells to the heterozygous forms that function normally. Unlike the therapeutic approaches envisioned for the treatment of bone marrow disorders, it appears to be improbable that cells can be harvested from the cystic fibrosis lung, genetically corrected, and returned to the host. The complex branching pattern of the lung would appear to preclude this approach. However, it is possible that DNA vectors can be delivered for correction directly to the lung by aerosol or direct installation. Two general approaches to correction can be envisioned: the so-called transient, or nonintegrative, expression of the CFfR cDNA; and integrative approaches. Each approach has its own virtues and is discussed separately.
Cystic Fibrosis Therapy in the 1990s
5.1 Nonintegrative Approaches In transient expression approaches, the cDNA coding for CFTR will be introduced into affected cells in vectors that do not require chromosomal integration for transcription. Typically, expression of the foreign protein by nonintegrative approaches will last from days to weeks. One approach envisioned is to utilise a cDNA cloned into a plasmid vector encapsulated in a Iiposome that can be delivered to the airway cell via inhalation. Liposomes can carry a variety of plasmid vectors, including vectors that can promote nuclear targeting of foreign DNA. Liposomal technologies for the efficient transfer of genes have improved rapidly, but at present they probably do not approach the degree of efficacy required to reach a significant fraction of airway epithelial cells, although it must be noted that the proportion of the epithelial cell population that must be corrected is unknown. It is also not yet known whether there will be a requirement for selective targeting of a liposome to one epithelial cell type or another. However, it is clear that cells facing the airway lumen will require correction. Nonintegrative therapy can also be delivered to the airway epithelial cells by a variety of viral vectors. Adenoviral vectors (Berkner 1988) are candidates for the treatment of cystic fibrosis lung disease becau,se of their tropism for airway epithelial cells (Castleman 1985). Recombinant adenoviruses with viral genes deleted and replaced with CFTR cDNAs are feasible. Similarly, it is possible that vectors that are continually maintained episomally [e.g Epstein-Barr virus vectors (Yates et al. 1985)] can be considered. Finally, the vaccinia viral system has been utilised to express CFTR transiently in airway epithelia cells and correct the Cl- channel defect (Rich et al. 1990); however, the fact that these viruses are ultimately lytic make them unlikely candidates for gene therapy purposes. 5.2 Integrative Approaches The alternative strategy in gene therapy is to utilise vectors that promote integration of the foreign cDNA into the host genome. The obvious ad-
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vantage of this approach is that the corrective gene can be maintained indefinitely in progenitor cells and can ultimately be viewed as effecting a 'cure' of cystic fibrosis airways disease. The efficient vector presently available for this purpose is the retrovirus. Retroviruses deliver RNA into the host cell that is reverse transcribed into DNA and, via retroviral integrase activities, incorporate the foreign DNA into the host genome. Recent studies have indicated that retrovirally delivered normal CFTR cDNAs correct the cystic fibrosis apical membrane Cl- channel in polarised airway epithelial cells (Olsen et al. 1991). This result, plus the observation that correction can be maintained in progenitor cells which can differentiate into transporting epithelial cells in vitro for up to 6 months, indicates that this may be an efficient means of treating cystic fibrosis lung disease. Nevertheless, major hurdles remain before this form of therapy can be considered for the treatment of lung disease. The first problem is efficiency. Retroviruses infect only proliferating cells. A low efficiency of retroviruses in gene transfer in the lung can be anticipated, because fewer than 1% of all airway cells are replication competent at any one time. The simplest strategy to offset this problem would be repetitive exposure to virus. Methods of boosting the size of the replication competent pool, perhaps in response to mild airway injuries, may be useful adjuncts to increase efficiency. A second major problem is safety. Retrovirally introduced genes integrate into the host genome at random. Positional factors may affect the rate of transcription which, if high, could have direct toxic effects on cell viability, e.g. via too many plasma membrane channels. Alternatively, random integration may interrupt important relationships between growth/antigrowth genes and potentially lead to a risk of carcinogenesis. Both problem areas need intensive investigation before this form of therapy is suitable for human trials.
6. Conclusions It appears that a variety of novel therapeutic agents will become available for the treatment of cystic fibrosis lung disease in this decade. It is clear
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that currently available anti-inflammatory drugs, and newly available agents that affect the composition of mucus, can be tested for efficacy in ameliorating preexisting lung disease. A new generation of agents that target the ion transport defects of cystic fibrosia airway epithelia will also become available. These agents offer the exciting possibility of not only arresting the progression of cystic fibrosis lung disease in patients with existing disease, but preventing the onset of disease in children treated early. It is conceivable that both agents which block excessive Na+ absorption (e.g. amiloride) and new agents which may directly correct the Cl- channel defect (triphosphate nucleotides) may be extremely efficient and potentially safe in this regard. Indeed, these agents alone may be able to perturb cystic fibrosis lung disease sufficiently to effect a functional ·cure'. Finally, it is possible that genetic techniques for attacking this disease will become important. Expression and delivery of normal recombinant CFTR protein may be feasible; alternatively, DNA therapy may prove practical. It has already been shown that expression of the normal CFTR cDNA will correct a phenotypic defect of cystic fibrosis in complex airway epithelial preparations in vitro. Access to the target tissue by the aerosol route makes delivery of the gene therapy vectors to the lung appealing. It is likely that transfer efficiency and safety will determine whether or not this form of therapy is brought to the cystic fibrosis arena in the next decade.
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Correspondence and reprints: Prof. Richard C. Boucher Jr, Pulmonary Division, Department of Medicine, CB# 7020, 724 Burnett-Womack Building, University of North Carolina at Chapel Hill, Chapel Hill, Chapel Hill, NC 27599-7020, USA.
