CF Clinical Research:

André Cantin, M.D

A Journey with a Destination

Cystic fibrosis is a chronic, often devastating disease in which most of the morbidity is associated with lung disease. Despite the major health problems that continue to afflict people who have cystic fibrosis, there is a growing number of highly productive young and less young adults who are proving to the world that this disease does not necessarily prevent one from reaching his or her destination.

It is the goal of clinical research to ensure that one day, all people with cystic fibrosis will make the journey towards their destination without worrying about the health consequences of cystic fibrosis.

Cystic fibrosis is a disease of tissues with tubular structures that are lined with an epithelium secreting protein-rich fluids. One of the more important proteins secreted in the fluids of diseased tissues is mucin. The organs that are classically involved in cystic fibrosis include the sinuses, airways, liver, pancreas, gastrointestinal tract and reproductive system. The cause of cystic fibrosis was identified by Doctor Lap-Chee Tsui of the Hospital for Sick Children of Toronto and his collaborators in 1989. Cystic fibrosis is caused by a single gene defect in the CF gene localized on chromosome 7. The CF gene codes for a protein known as the cystic fibrosis transmembrane conductance regulator or CFTR. The CFTR protein normally is inserted in the surface membrane of epithelial cells that line mucous membranes. The function of CFTR is to control the flow of negatively charged ions (anions), particularly chloride and bicarbonate. Regulation of ion traffic through mucous membranes plays a key role in determining the viscosity of mucus.

Several classes of gene mutations have been identified in subjects who have cystic fibrosis. The gene mutations associated with the most severe forms of cystic fibrosis are most often found in classes I, II and III, while gene mutations of classes IV and V are often associated with mild clinical manifestations. Class I mutations result in the absence of transcription of the gene and therefore the absence of CFTR protein. Class II mutations represent the most frequent mutations seen in cystic fibrosis populations of northern Europe and North-America and include the DF508 amino-acid deletion. This mutation results in a misfolded CFTR protein which is degraded before most of the protein can travel to the mucous membrane. Class III mutations affect the regulation of the opening and closing of the CFTR anion channel while class IV mutations affect the ion selectivity of this channel. Finally, the class V mutation result in a normal CFTR protein that is produced in lower quantities and is associated with a milder disease that is sometimes manifested only by infertility in men.

"The principal functions of CFTR are to secrete chloride…"

The principal functions of CFTR are to secrete chloride, bicarbonate and possibly a third organic anion known as glutathione. Glutathione is a relatively large molecule compared to the other ions and its main characteristic is the presence of a sulfhydryl (SH) group which gives it antioxidant properties and allows it to break up disulphide bonds. Bicarbonate is an essential ion for the maintenance of normal pH, and its absence is associated with acidification of fluids at the surface of mucous membranes. Finally, chloride transport controls the quantity of salt and water in the fluid at the mucous membrane surface.

The most important protein in the regulation of mucus viscosity at the surface of mucous membranes is mucin. Mucin is a protein to which is bound a very large amount of sugar. The amount of water surrounding the sugars within the mucin structure plays a key role in regulating the viscosity of mucus. In addition, mucin has many disulphide bonds which can increase mucus viscosity. Finally, mucin viscosity is highly dependent on pH and the acidification of mucin-containing solutions results in an increase of viscosity. It is therefore thought that the major functions of CFTR which are regulation of salt, water, glutathione and bicarbonate secretions may play key roles in determining the visco-elastic properties of mucin. However, it is essential to note that mucus in older children and adults with cystic fibrosis contains many molecules others than mucin and therefore the modulation of airway secretion viscosity by water, glutathione and pH may not be the same as is observed with pure mucin in a test tube.

"…cystic fibrosis pulmonary disease is caused by a severe defect
in mucus clearance from the airways."

Investigators from Doctor Jeffrey Wine's laboratory in California have recently determined that mucin-rich secretions from sub-mucosal glands of the airways seem more viscous in the absence of expression of CFTR. It is interesting to note that the cells expressing the highest concentrations of CFTR in the sub-mucosal glands are those that secrete the most water. Similar cells are also found in smaller amounts at the surface of airway mucous membranes and in other mucus secreting tissues. Doctor Richard Boucher's group in North Carolina has recently demonstrated that the liquid at the surface of the airways in which there is deficient CFTR expression is markedly dehydrated. This is associated with the formation of very thick sticky mucus plaques that can be identified in the airways of subjects with cystic fibrosis. Based on these observations and those of many other investigators over the past two decades, there is now a widely accepted view that cystic fibrosis pulmonary disease is caused by a severe defect in mucus clearance from the airways. It is likely that this mucus clearance defect is also associated with altered local host defences, both of which lead to the accumulation and proliferation of bacteria at the airway surface. The presence of bacteria rapidly triggers an inflammatory response characterised by the migration of white blood cells known as neutrophils to the airway surface. The airway neutrophils release toxic products in an attempt to kill bacteria; however, for reasons that are not fully understood, this inflammatory response does not succeed in killing bacteria. The inflammatory response becomes excessive leading to tissue destruction not only by bacteria but also by products from the white blood cells themselves. One of the major damaging products released by white blood cells is an enzyme known as elastase which can degrade airway tissues, increase mucus secretion and attract more of the damaging white blood cells. In addition, elastase strips receptors on white blood cells and proteins on bacteria resulting in defective ingestion of bacteria by the white blood cells.

In recent years, clinical research has been based on our understanding of the pathophysiology of cystic fibrosis and has resulted in changes to our practice. Three examples of changes in our clinical practice that have directly resulted from clinical research in cystic fibrosis are the use of aerosolised antibiotics, aerosolised DNase and the care we now give to the prevention of acquisition of certain bacteria, particularly of the B. cepacia complex. Although these changes in clinical practice have definitely resulted in improvement for many persons with cystic fibrosis, much still has to be done. Several novel therapeutic approaches are being studied in clinical research programmes.

"The therapeutic approaches currently being investigated…"

The therapeutic approaches currently being investigated address all aspects of cystic fibrosis pathophysiology including the correction of the abnormal gene and the abnormal protein, the improvement of mucous membrane ion transport, the better treatment of lung inflammation, infection and tissue destruction and ultimately the replacement of organs that have failed by transplantation.

Correction of the abnormal gene has been an area of intense investigation over the last 10 years. Major problems have occurred in 2 related areas of gene therapy development. First, transfer of the normal gene to disease cells has often been inefficient and when efficiency was improved, the approaches were associated with a very short duration of expression of the normal gene. This lack of sustained gene expression was associated with a second major problem, that of an inflammatory reaction initiated by the vector used to transfer the normal gene to CF tissues. The inflammation associated with certain types of vectors not only reduces the duration of gene expression but can also be life-threatening. These very important problems recently led to a major reassessment of all the gene therapy clinical protocols by investigators, universities and regulatory agencies. Although many major changes have occurred in approaches to gene therapy for cystic fibrosis, the field is still very much alive. One of the more promising developments that is currently being explored involves a transfer vector known as the adeno-associated virus (AAV). The AAV vector is not known to produce disease in humans and is not associated with an inflammatory reaction but does effectively transfer genes to epithelial cells. A phase I study in CF patients showed that using this vector, the normal CF gene can be safely delivered to epithelial cells in the sinuses, and this results in an improvement of chloride secretion by the cells. A phase I aerosol study, using the same approach, also demonstrated that the treatment is well tolerated and, interestingly, the gene was expressed for ninety days at the highest dose used in this study. The company Targeted Genetics is currently working with the Cystic Fibrosis Foundation in the United States to do more research in the development of this gene therapy approach. A phase II study has been initiated in 36 patients to determine the effect of this therapy on lung function and sputum bacteriology.

Another approach to the gene defect that is relevant to those patients with class I mutations is the use of drugs that allow the transcription of the gene to occur despite the presence of a mutation. This approach is only valid for the 10% of cystic fibrosis patients who have a gene mutation resulting in the lack of transcription of the gene. Some populations of patients with cystic fibrosis such as those from the Ashkanazi Jewish population express a much higher incidence of this type of gene mutation and one of the promising approaches that may help these people is the use of aerosolised gentamicin. The topical administration of gentamicin to the nasal surface has been shown to correct chloride transport in cystic fibrosis patients with this class I mutation. Currently, clinical trials of this approach are ongoing in Israel and at the University of Alabama.

In addition to therapeutic developments targeting the gene, many groups are now studying approaches that directly address the CFTR protein. The most common mutation leading to cystic fibrosis is the class II DF508 mutation which results in a very poorly folded protein which cannot travel to the cell surface where it normally functions. Doctor Pamela Zeitlin of the Johns Hopkins University in Baltimore has demonstrated in recent years that it is possible to treat epithelial cells containing this common mutation with drugs that allow the mis-formed protein to travel to its normal location and resume at least part of its normal function. Doctor Zeitlin has now completed a number of early clinical trials in CF patients and has shown that treatment with sodium 4-phenylbutyrate is well tolerated by patients and currently her group as well as a group at the University of Pennsylvania are exploring the usefulness of this drug in larger clinical trials.

In addition to approaches that mobilise CFTR protein to the membrane surface, several groups are exploring the possibility of using other drugs that would activate the small amounts of CFTR protein that are still present in mucous membrane of patients with cystic fibrosis. The logical assumption of this approach is that if one can markedly increase the function of the few remaining CFTR proteins, then the manifestations of cystic fibrosis may be decreased. It has been shown that several drugs including CPX (8-cyclopentyl-1-3-dipropylxanthine), genistein, and a novel class of drugs known as 7,8-benzoflavones can directly activate the defective CFTR protein, thus restoring at least part of its anion transport properties. Investigators at the Children's Hospital of Philadelphia are currently exploring an approach using both genistein and sodium 4-phenylbutyrate in an attempt to increase the amount of CFTR protein as well as its function in CF patients.

Although the activation of defective CFTR protein may prove successful, several investigators have opted for an alternate approach in which anion channels not related to CFTR are being recruited and activated using different novel drugs. There exists a class of chloride transporting proteins known as calcium-dependent chloride channels which may, if the theory is correct, be able to replace the deficient CFTR function. Investigators from the University of North Carolina have developed molecules that stimulate these different chloride channels. A clinical study, using one of these molecules known as INS365, has recently been completed and demonstrated that the drug is safe. However, the drug was rapidly degraded in the lung and these investigators are now doing clinical studies on a more stable derivative of the same drug class. This new derivative is known as INS37217. Other investigators working with the company known as MoliChem Medicines Inc. are exploring the potential usefulness of Moli1901 also known as Duramycin to enhance chloride transport through cystic fibrosis epithelium.

Clinical research is also being conducted to find agents and approaches that will improve mucus clearance from the airways. One product known as Dextran is being developed by investigators in Canada as a possible modulator of mucus visco-elastic properties. A phase I trial of aerosolised Dextran has just been completed in Toronto in normal subjects. The animal studies and the phase I trial in normal subjects suggest that the product is safe and a phase II trial is currently being planned to test Dextran aerosolisation in patients with cystic fibrosis. An alternate approach to improving mucociliary clearance is to use modern technologies applied to techniques of physiotherapy. In particular, there have been a number of developments with high frequency chest wall oscillation devices and investigators are now exploring the potential benefits of mucociliary clearance vests alone or in combination with inhalation therapy. These are multicentered trials that should provide clear information on the optimal and most convenient physiotherapy techniques for mucociliary clearance in cystic fibrosis.

"A large Pseudomonas aeruginosa vaccine trial is near completion…"

Several approaches are being explored for more effective anti-infective therapy and prevention. A large Pseudomonas aeruginosa vaccine trial is near completion in Europe and results should be available within one year. In addition, one of the more intriguing and promising studies currently being conducted both in the United States and in Europe explores the potential usefulness of macrolide antibiotics for the treatment of cystic fibrosis patients chronically colonised with Pseudomonas aeruginosa. Macrolide antibiotics are not good Pseudomonas aeruginosa antibiotics based on traditional microbiological studies. However, several microbiology groups have demonstrated in recent years that macrocodes can affect the expression of important genes by Pseudomonas aeruginosa and may actually play a very positive role in diseases characterised by a slow growth of these bacteria in a coating known as bio-films. Phase III trials of macrolides in cystic fibrosis patients are underway and the results of these should be available before the next North-American Cystic Fibrosis Conference in 2002.

"…natural host defences may be improved using novel therapies."

In addition to approaches using antibiotics to treat lung infection, one of the more intriguing and potentially interesting concepts is that natural host defences may be improved using novel therapies. For example, a product normally secreted by our white blood cells is g-interferon (gIFN). We know that gIFN can increase the capacity of white blood cells to defend us against various organisms including viruses and bacteria. gIFN has now been cloned and purified and has been shown to help certain populations of patients who have chronic infections. A clinical research study is now underway to explore the potential benefits of gIFN treatment in cystic fibrosis. Another approach that attempts to enhance natural host defences is the use of a1-antitrypsin which is a safe, well tolerated inhibitor of white blood cell elastase. a1-antitrypsin aerosol is limited by the large amounts of protein that must be aerosolised in order to effectively suppress all lung elastase in CF patients. However, recently PPL Therapeutics and Bayer have formed a partnership that has allowed the production of large amounts of human a1-antitrypsin in the milk of sheep. This technology has allowed the production of sufficient amounts of the protein to complete a multicenter trial of aerosolised a1-antitrypsin in CF patients. The results of these studies are now being collated and analysed and should be available within the next few months.

Another inhibitor of elastase that has been developed by Doctor Eileen Remold and her colleagues at Harvard University is a protein known as monocyte neutrophil elastase inhibitor. Tests performed in test tubes and in animals have shown excellent inhibition of elastase and a good safety profile. It is likely that in the near future we will have much more information on the potential usefulness of elastase inhibition as a therapeutic approach in cystic fibrosis lung disease.

"…without the concern of having cystic fibrosis disrupt their journey."

In summary, the discovery of the CF gene and its protein have led to a tremendous improvement in our understanding of the pathophysiology of cystic fibrosis particularly as it affects the lung. We are now seeing concrete evidence of the benefits of this improved knowledge in the form of novel applied therapies that are being explored in well-structured clinical trials. It is clear that not all of these approaches will be successful but it is just as clear that if we do not actively support clinical research in cystic fibrosis, no new clinical approaches can be developed. I believe that we are making significant progress in reaching the goal that we have established collectively. This goal is to ensure that all people living with cystic fibrosis can reach whatever destination they choose without the concern of having cystic fibrosis disrupt their journey.