August 13, 2012

Cystic Fibrosis Research Gains Steam
By Carolyn Gutierrez
For The Record
Vol. 24 No. 15 P. 22

By homing in on the disease’s source and studying the aggressive use of antibiotics in its treatment, the medical community believes it’s making inroads toward better care.

Cystic fibrosis (CF) is a genetic disease caused by a defective protein that disrupts the salt and water content of the mucus lining of the lungs, pancreas, and other organs. Instead of producing a thin, slippery layer of mucus to hydrate and protect the organs, the protein, known as the cystic fibrosis transmembrane conductance regulator (CFTR), generates a thick and sticky mucus throughout the body, clogging the lungs and impeding normal gastrointestinal activity. Lung function is severely compromised in most CF patients as the sticky mucus causes labored breathing and a sustained vulnerability to infection.

About 30,000 children and adults in the United States have been diagnosed with CF. Approximately 10 million Americans are carriers of the CF gene but do not have the disease. When each parent carries one copy of the defective gene but neither has the disease, there is a 25% chance of their child inheriting both copies and developing CF; a 25% chance of not getting the disease or carrying the CF gene; and a 50% chance of being a carrier, inheriting one copy of the gene.

Although there is no cure for CF, because there are more than 1,000 mutations of the gene, there is a wide range of severity, making it difficult to predict a patient’s life expectancy. Still, the average life expectancy for CF patients has risen dramatically over the past 15 to 20 years. The Cystic Fibrosis Foundation’s national patient registry contains anonymous data on more than 26,000 CF patients in the hopes that a vast network of information can bring patients and researchers closer to a cure.

“The patients are living longer,” says William B. Guggino, PhD, director of the CF research development program at Johns Hopkins Cystic Fibrosis Center. “In the 1940s and 1950s, children with CF used to live to be 5 years old and now the average age of survival is 40. So it’s gone up, and that’s primarily by treating the symptoms.”

Antibiotics, anti-inflammatory drugs, mucus thinners, and bronchodilators can help prolong the lives of CF patients. Because food does not move down the gastrointestinal tract effectively and enzymes are unable to travel from the pancreas into the stomach, most CF patients need to take pancreatic enzymes along with implementing specialized nutritional management.

Since there is a malfunction in how the CFTR protein channels salt out of the body, one of the classic CF symptoms is “salty” skin. “Chloride is high in these children,” Guggino says, “so Grandma would say, ‘The baby tastes salty when I kiss him.’ When we exercise, it’s normal for us to be salty when we sweat. Cystic fibrosis patients are salty all of the time because they have this defect in their CFTR.”

Other possible symptoms indicating CF in a child may be persistent coughing, wheezing, frequent lung infections, greasy or bulky stools, and a general failure to thrive, including poor growth and malnutrition.

Most children with CF are diagnosed before they reach the age of 2. Measuring the amount of chloride in their sweat has long been the gold standard for CF testing, but genetic testing using a blood sample or a sample of cells from inside the cheek has recently provided another option. Children who are diagnosed early have a greater chance of long-term survival because pulmonary function can be monitored and treated before extensive colonies of bacteria set in. Also, families can learn important nutritional strategies and techniques to help the child clear his or her airways.

Current Research
Although scientists cannot yet predict when there may be a cure for CF, researchers are homing in on the very source of the harrowing disease: the defective CFTR protein.

A research team at Ryerson University in Toronto is synthesizing molecular probes to study the CFTR protein in the hopes that better drugs can be developed to make repairs. The research team, led by Russell Viirre, PhD, a synthetic organic chemist and assistant professor in Ryerson’s department of chemistry and biology, has partnered with the Bear group at The Hospital for Sick Children.

“We do all of the chemical synthesis in my lab, so we are making these tools,” Viirre says, “but it’s Dr Christine Bear and her group who actually do the experiments with the protein. It’s a real collaboration. They have the expertise to handle and do experiments with the protein; we have the expertise to make molecular probes. These molecular probes are tools that otherwise wouldn’t exist for protein researchers like the Bear group to try and pinpoint exactly where these small molecules bind to the protein.”

Vertex Pharmaceuticals has developed several CF drugs that show great promise. However, since there are a vast number of possible mutations in the CF gene, the drugs can address only very specific mutations.

Viirre compares the defective protein to a large, complicated lock, with the drug acting like a key that may fit. For the past two to three years, the team has been studying two previously tested CF drug compounds. Making derivatives of these drugs to test various interactions, the research team is hoping to pinpoint where the CFTR protein interacts with a given drug—essentially trying to find a “keyhole” on the protein. By narrowing down where on the protein the small molecule binds to cause restoration of function, the scientists can then modify the drugs for further testing.

“These drugs—or actually we tend to refer to them as small molecules—seem to essentially restore the activity of the defective protein in vitro,” Viirre says. “What we don’t know is how they do it. And so the question is, what is it that the small molecule does to the large one—the protein—that suddenly restores its normal function?”

The researchers use a handful of different approaches when testing the molecular interactions. For example, in one test, the small molecule may form a covalent bond to the protein, while in another test the small molecule may fluoresce a different color when it’s close to the protein. Ultimately, Viirre and his team are looking to see whether the drug molecules all interact with the protein at the same spot.

“In other words,” Viirre says, “are they all keys for the same keyhole or are there many different keyholes on the protein? This would help us to understand exactly what sorts of things we can do at the molecular level to rescue the activity of this protein. If we understand exactly what that interaction looks like, then we can make the drug that is perfect for that interaction—the strongest and most potent drug to restore the activity of the defective cystic fibrosis protein.”

Antibiotics’ Role
Perhaps the most menacing health concern for CF patients is a bacterial infection in the lungs. Lung damage from frequent infection is the most common cause of mortality for those with CF. The current treatment standard is aggressive use of antibiotics to keep infection at bay.

Working within the context of the Human Microbiome Project—a “road map” initiative established by the National Institutes of Health that analyzes and characterizes microbial communities throughout the human body—a team of researchers from the University of Michigan performed a longitudinal study of the bacterial communities in the airways of six adult CF patients.

In the 10-year study, the researchers found that aggressive use of antibiotics led to a decreased diversity of bacteria in patients’ lungs. Essentially, this lack of diversity enabled the treatment-resistant bacteria to thrive in the lungs as their competition was eradicated. Of the six patients followed, three had fairly stable lung disease over the 10-year period, and three had the more typical course of lung disease for CF: steady, severe loss of lung function.

 “What we found was that in the three stable patients the diversity of bacteria in their lungs remained pretty constant; it didn’t fluctuate that much,” explains study author John J. LiPuma, MD, the James L. Wilson, MD, research professor of pediatrics and communicable diseases at the University of Michigan Medical School, who has been studying CF lung infections for 25 years. “They continued to have quite a variety of different bacteria in their lungs. In the three patients who had a more progressively downward course, what we found was that their communities became less and less diverse over time.”

The scientists discovered that the diversity of bacteria decreases significantly in patients in advanced stages of the disease. Fewer sets of bacteria were recovered from their lungs: two or three as opposed to the few dozen that had been found in the early stages of their disease.

The few surviving species of bacteria in the lungs of the extremely ill CF patients were found to be extraordinarily stable and did not seem to be affected by antibiotics. In looking at long-term antibiotic management, the Michigan researchers recognized a pattern emerging: Colonies of lung bacteria were diverse when the patients were younger adults but, over time, that diversity narrowed as the patients’ disease worsened and antibiotic use increased.

“We did some statistical analyses to try to understand what the main driver of this decreased diversity was,” LiPuma says, “because what we had were associations. We had an association between age and decreased diversity, an association between decreased lung function and decreased diversity, and an association with increased antibiotic use and decreased diversity. So when we did some statistical analyses to control for antibiotic use, then the effect of age became insignificant. And when we controlled for antibiotic use, the effect of lung function became insignificant. These results suggest that antibiotics are the main driver of decreasing diversity.”

Because the study sample was small, LiPuma hopes expanded studies can be performed to determine whether the results hold with larger numbers of CF patients.

What LiPuma’s research means for the future of CF care is uncertain. “What we do know is that antibiotic therapy has absolutely led to increased survival in CF,” he says, “and there’s plenty of data to not only suggest but to indicate that the more intensive antibiotic use over the past decade has benefited patients greatly and that early aggressive therapy now for new infections is holding real promise for either eradicating early infection or at least postponing it and thereby preserving lung. What this study does is it raises the question, is there a tipping point after which we are starting to work against ourselves, in a way, with antibiotics, and if so, how is that happening?”

One hypothesis, according to LiPuma, is that antibiotics may be killing off some species that are helping to keep other, more virulent species in check in the lung. Perhaps more judicious use of antibiotics after a certain point in time in the disease progression is needed. The study makes no recommendations with regard to care for CF patients because the answers remain unclear.

“What we do have, though,” LiPuma says, “is a better understanding of what’s happening in the lungs of patients through the course of 10 years, watching their disease progress. As we understand it more and confirm some of the findings found in this paper, it then generates hypotheses that can be tested, such as maybe we ought to think about a slightly different way of giving antibiotics. The study also raises possibilities about mechanisms: What’s the mechanism by which these last few surviving species are able to hang on, are able to fill the room we’ve made by clearing out some of the other ones? So it’s informative because it gives us a clearer picture of what’s going on and whenever we have a clearer picture, it generates more questions. Those questions can generate hypotheses that may eventually, hopefully, lead to interventional trials.”

— Carolyn Gutierrez is a freelance writer based in New York City.