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Cover Story

September 1, 2008
Volume 86, Number 35
pp. 15-23

Breathing Easier

Cystic fibrosis patients could soon benefit from breakthrough small-molecule drugs that treat their underlying disease instead of just their symptoms

Lisa M. Jarvis

ON A TYPICAL school morning, Jeanmarie Youngblood wakes up at 4:45 AM to start her daily routine, an hour and 20-minute ordeal that doesn't even include a bowl of cereal. If this was a case of typical teenage-girl vanity, she might be spending the time showering, blow-drying her straw-colored hair, and picking out the right pair of skinny jeans. But for Youngblood, setting her alarm for that bleary-eyed hour isn't about looking good in homeroom. It's about getting her lungs in shape for the day.

Vertex Pharmaceuticals
BREAKTHROUGH Scientists at Vertex Pharmaceuticals have discovered drugs to treat the genetic defect that causes cystic fibrosis.

Youngblood has cystic fibrosis, a disease that causes thick, sticky mucus to clog her lungs and disrupt her digestive tract. It's a trait she shares with some 30,000 other Americans who inherited a copy of an errant gene from both their mother and father.

The first order of business when Youngblood stumbles down the stairs of her Brooklyn, N.Y., home is to attach a hose to her nebulizer, a device that aerosolizes a drug, and then load it with albuterol, which opens the airways in her lungs. Next, she puts on a specially designed vest that compresses and releases her chest wall. Looking like a life jacket, it gives a vigorous massage that helps loosen the mucus in her chest and keeps an otherwise persistent cough at bay. For the 30 minutes she wears the vest, she inhales hypertonic saline, a recent addition to the routine that helps to thin and clear mucus.

After that, Youngblood changes the hose on her nebulizer yet again to inhale Tobi, an aerosolized version of the antibiotic tobramycin. Another hose change, and she breathes in Pulmozyme, an enzyme that snips the extracellular DNA in the lungs' mucus, an additional method of thinning everything out.

She repeats the entire routine, without the Pulmozyme, around dinnertime. Keeping up with the nearly three-hour-per-day program is no small feat. "It's a lot of time I spend sitting, not being able to do things," Youngblood concedes. Yet it is also a fact of her teenage life, something that she has to do to feel okay.

Cystic fibrosis boils down to a hydration problem. The mucus lining our lungs, just like the stuff that lines our nostrils, acts like flypaper, catching bacteria, dust, viruses, and other particles we inhale. The cilia then sweep that mucus out, a process we see in action when we have a cold.

But the level of hydration needs to be just right to keep the mucus flowing. There is a delicate balancing act going on inside our lungs: Salt is being absorbed and secreted to draw water in and out, maintaining a consistent thin layer of fluid on our airways.

That balance is off in people with cystic fibrosis. They all have a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which carries the instructions for making a protein that shuttles chloride ions from one side of a cell membrane to the other. CF patients absorb too much salt and have trouble secreting it; as a consequence, their lungs are dotted with dried-out pockets that get clogged with thick mucus. The pockets are a breeding ground for bacteria. Patients are afflicted with chronic infections, most commonly caused by a nasty bug called Pseudomonas aeruginosa.

"The ultimate irony is that in your lungs right now you have about 10 mL, or one-third of a teaspoon, of water on your airways," says Richard Boucher, director of the Cystic Fibrosis Pulmonary Treatment & Research Center at the University of North Carolina (UNC), Chapel Hill. "In CF, at least early in life, you have almost none." All that doctors are trying to do throughout the course of a CF patient's life is replenish that tiny bit of fluid and keep it there.

IT'S A TASK that is much easier said than done with existing treatments. To date, patients have been stuck with drugs that treat their symptoms rather than the underlying disease causing them. Youngblood's regime, for example, is mostly palliative: The goal is to keep her Pseudomonas infection from getting out of control while breaking up enough mucus in her lungs so she can breathe easier and cough less often.

8633cov3_SiGNacxd2.tif CF Foundation
BREATHE IN CF patient Zach Berkley uses a nebulizer as part of his therapy.

But in the next few years, a stream of new small-molecule drugs aimed squarely at repairing the hydration balance could reach patients. The drugs offer the potential to not only improve the health of CF patients like Youngblood but also dramatically change the outlook for people newly diagnosed with the disease. And because some of the drugs are in pill rather than aerosol form, they could make life a lot easier for patients.

This promising pipeline would not exist without the ambitious efforts of the Cystic Fibrosis Foundation, which has funded much of the discovery and development of those products through its investment arm, Cystic Fibrosis Foundation Therapeutics. The nonprofit organization provides incentives to companies to develop products for a relatively small group of patients who seek newer and better drugs (C&EN, May 7, 2007, page 19).

The need for better drugs is acute. It is virtually impossible to get rid of bacteria once they take up residence in a patient's lungs, says Bob Beall, president of the CF Foundation. Their presence sets off a vicious, lifelong cycle of treatment for patients. "They use antibiotics, the infection comes back; they use them again, it comes back again," Beall says. "Eventually, every infection takes its toll on lung function. Eventually, most patients die of lung destruction."

That harsh reality is why patients like Youngblood work so hard to keep those infections under control. Even with her daily routine, Youngblood spends a few days in the hospital each year as a housekeeping exercise to clear out a persistent Staphylococcus infection. Before Tobi was introduced in 1998, Youngblood had to make additional hospital visits to treat her Pseudomonas infection.

Furthermore, CF is not only a problem of the lungs. That sticky mucus blocks the movement of enzymes in the pancreas, making it difficult for CF patients to absorb nutrients; eventually, pancreatic function breaks down entirely. So in addition to her twice-daily lung-treatment routine, Youngblood swallows five pills containing enzymes at every meal or snack to help her intestines absorb much-needed nutrients. She also takes multivitamins, salt pills, and the inhaled asthma treatment Singulair daily and the antibiotic Zithromax three times per week.

Such routines have been effective. When the CF Foundation was established in 1955, patients barely lived to school age. By the 1980s, with more antibiotics available, life span was extended to the late teens. Today, because of more effective drugs, patients generally live to be older than age 37.

The drugs offer the potential to not only improve the health of CF patients but also dramatically change the outlook for people newly diagnosed with the disease.

But the foundation thinks it can do much better. "We've always felt that if we could find out why those thick, tenacious secretions are building up, we could stop that buildup, thin out the secretions, and maybe bacteria won't even want to live there," Beall says. "If we could treat the basic defect, we could have a better potential for treating the disease."

The CFTR gene was discovered in 1989, but it took years of research to unravel how it was actually causing the disease. "It wasn't really until we developed the systems that allowed us to understand that these CF patients were missing this thin film of liquid that it really became obvious how the whole pathogenesis of CF evolved," UNC's Boucher says.

Armed with that information, drug companies are taking two approaches to addressing the underlying disease. One camp is trying to correct the genetic defect—that is, to actually manipulate the formation of the CFTR protein to render it functional. The other camp is working on opening alternative routes for transporting salt to the lining of the lungs and intestines. Beall says both show promise and even have the potential to be used together for maximum effect.

Vertex Pharmaceuticals has been working with the CF Foundation on figuring out how to fix the broken chloride channel for nearly a decade. In the late 1990s, the nonprofit enlisted Aurora Biosciences, which was later acquired by Vertex, to develop functional assays that could be used to find molecules that corrected CFTR function.

The project has focused on multiple ways of repairing the errant protein. Because CFTR's main function is to move chloride ions from inside the cell to the outside, it can be thought of as a gate, says Paul Negulescu, vice president of research at Vertex. Patients have different kinds of problems with that gate: Most commonly, they don't have enough gates, but the gates can also be in the right place but "rusty" and not opening properly.

Vertex developed assays to look for compounds that increase the number of gates on the cell surface—drugs known as "correctors"—and compounds that could increase the flow of chloride through existing gates, or "potentiators."

At the time, "we didn't have a real chemical notion about what type of molecule it would take to fix the CFTR," Negulescu says. "The philosophy was, 'the bigger the library, the better.' " The assay took about a year to develop, and for several years the company screened, rescreened, and counterscreened to try to find hits. Around 2003, molecules finally started showing up that looked like they could be developed into drugs.

Part of the challenge was that Vertex focused on finding orally available drugs to correct the genetic defect. Taking a pill would not only reduce the burden on patients who already take a lot of inhaled drugs but could also have a systemic effect. "In the ideal world we would be able to treat all the organs affected by CFTR dysfunction, not just the lungs," Negulescu says.

Image Title Courtesy of Jeanmarie Youngblood

The CF Foundation has sunk about $80 million into the collaboration with Vertex and is now starting to see the fruits of its investment. After several years of optimizing the leads found in the assay, the company has sent two compounds to the clinic. Phase II trials of VX-770, a potentiator, showed that after just two weeks of treatment, lung function improved 10% in a small population of patients with a specific CFTR defect.

The second part of the trial will look at how patients fare after 28 days; pending the results, Vertex believes it could initiate a registration program in 2009, but it also plans to look at how the drug works in patients with the most common CFTR defect, the ΔF508 mutation. Meanwhile, VX-809, a corrector, is in Phase I studies to establish safety and proper dosing.

THE COMPANY is still trying to figure out how the drugs correct the defect. "It's a challenge to understand at the protein level exactly what these compounds are doing," Negulescu acknowledges. "We do know for sure that VX-770 increases the flow of ions through the channel."

Still, the discovery of those molecules has helped researchers gain a better understanding of how the disease works, UNC's Boucher says. "With the Vertex compounds, we're learning how the CF protein matures within the cell," he says. Scientists now have better information about how the protein folds, what partners it uses to fold, and where it interacts with itself.

Although the Vertex compounds may be useful for the majority of CF patients, smaller patient populations have various other CFTR mutations that also need better medicines. PTC Therapeutics is looking to repair a "nonsense" mutation carried by a small subset of CF patients. Normally, a ribosome attaches onto the "start" site of messenger RNA (mRNA) and reads through instructions for building the CFTR protein until it reaches a specified "stop" site. In CF patients with the nonsense mutation, a premature stop signal on the mRNA causes the ribosome to fall off before the full protein is built.

Gentamicin and other aminoglycoside antibiotics have already been shown to allow the normal CFTR protein to be built, explains Langdon Miller, PTC's chief medical officer. However, those bulky semisynthetic molecules won't suffice as treatment for a chronic condition; they aren't potent enough to treat CF, are toxic, and have to be given intravenously or by injection.

PTC was looking for potent, synthetic molecules that could be taken as a pill. The company screened some 600,000 compounds for ones that would allow ribosomes to make full proteins. Researchers had a ready check in their assay for whether the molecules were making the full-length proteins; they inserted the instructions for the firefly enzyme luciferase into the cells. Compounds that were active in the screen would then also cause the cells to make luciferase; in other words, the cells would simply light up when the functional CFTR protein was present.

After making several thousand analogs based on the screening hits, PTC scientists homed in on PTC124, which seems able to turn that misplaced stop sign into a yield signal. Even though its exact mechanism is unclear, Miller says the compound appears to bind to the 28S ribosomal subunit and induce some conformational change in the ribosomes that enables the full translational process to proceed.


Early trials of PTC124 were so promising that Genzyme recently agreed to pay $100 million for access to the drug, which is also being tested to treat Duchenne muscular dystrophy (C&EN, Aug. 25, page 17). PTC is poised to launch a Phase IIb study designed to collect enough evidence to ask the Food & Drug Administration for approval. If all goes well, the drug could hit the market by 2011.

While Vertex and PTC are working to directly repair the specific defects of CFTR that cause a roadblock to chloride transport, other companies are exploring side roads to hydration. Two alternative routes are being exploited: turning on the calcium-dependent chloride channels to shuttle more ions to the surface and blocking the epithelial sodium channel to create an osmosis-driven pump that draws liquid to the surface of the lungs and, consequently, sweeps away mucus.

Inspire Pharmaceuticals' lead candidate, denufosol, had its origins at UNC Chapel Hill, where a group of researchers, including Boucher, discovered that modulating the P2Y2 receptor in the lung stimulated the calcium-dependent chloride channel while inhibiting the epithelial sodium channel. The UNC researchers also figured out that those pathways are naturally modulated by uridine triphosphate (UTP), which pushes more chloride ions through the surface of the lungs while keeping water from being reabsorbed.

But UTP didn't make sense as a drug, says Benjamin R. Yerxa, chief scientific officer of Inspire. The terminal phosphate makes it vulnerable to hydrolysis, both chemically and enzymatically; it has to be kept frozen as a solid or refrigerated as a liquid. Those restrictions simply weren't realistic for patients with a chronic disease.

Inspire started making analogs of UTP and eventually discovered a class of compounds called dinucleotides that are just as potent as the naturally occurring compound but much more stable. The company's first dinucleotide, diuridine tetraphosphate, showed potency equal to UTP and was stable at room temperature. "UTP has to be frozen at –78 ºC," Yerxa notes. "It took me six months to believe I could keep diuridine tetraphosphate on the benchtop."

That was a start because it meant that drugs could be transportable and long-lasting. Yet to successfully treat CF, a drug also needed a long duration of action. Our lungs are supercharged with enzymes to battle inflammation and infection; to make a good drug, a dinucleotide would need to survive that pulmonary soup long enough to get the chloride flowing.

The company finally struck upon denufosol, an asymmetric molecule with a uridine on one side and a deoxycytidine on the other. Yerxa says this off-balance form seems to confuse the enzymes, and the compound remains active in the lungs for up to eight hours.

The CF Foundation helped to fund a Phase II trial of the drug. According to Yerxa, the backing removed a lot of the risk for a small company deciding where to spend limited capital. The beauty of denufosol, Beall says, is its ability to work in any CF patient, regardless of his or her genetic mutation, because it is tapping into those alternative chloride channels. He says the foundation is encouraged by results from the first Phase III trials showing that lung function improved after 24 weeks on the drug. Inspire is currently enrolling patients in a second Phase III trial and expects results next year.

ALTHOUGH PROMISING, denufosol has its downside. It has to be taken three times a day through a nebulizer, not an inconsequential time commitment, though not far beyond patients' current regimen. "That midday dose can be a challenge," Yerxa concedes. "But the parents and families are so dedicated to the overall health and treatment of their kids that they are extremely compliant."

Parion Sciences is also trying to restore salt transport in the lungs. In 1999, UNC's Boucher asked the then-retired biotech industry veteran M. Ross Johnson to get back into the lab and try to design drugs to block absorption of water by inhibiting the epithelial sodium channel. With a small grant from the CF Foundation, Johnson found such promising compounds in his initial work that the two scientists started a company.

They were looking for small molecules, recognizing that their challenge was the likelihood that activating the epithelial channel would cause a buildup of calcium in the kidneys. Parion needed to design a molecule that could either be directed away from the kidneys or be metabolized into compounds that would be less active if they reached the kidneys, says Johnson, now Parion's chief executive officer.

Parion ended up with a series of 2-substituted acylguanidine analogs of amiloride. The two lead candidates, P-522 and P-680, could be administered in conjunction with hypertonic saline, an existing treatment that sucks water into secretions to hydrate the tissues. The Parion drugs would help keep that water from being reabsorbed, Boucher says.

Parion has licensed the drugs to Gilead Sciences, which became active in CF research through its 2006 acquisition of Corus Pharma. P-680, now called GS 9411, is in preclinical trials. Gilead is awaiting FDA's approval for a former Corus drug, an aerosolized version of an antibiotic that had previously been administered only intravenously. That same formulation expertise could be applied to the Parion product.

Although it is backing a pipeline of drugs that have the potential to dramatically improve the lives of CF patients, the CF Foundation shows no signs of slowing its efforts to find and fund better medicines. Its ultimate goal is to improve patients' lives while also reducing the treatment burden. One way to accomplish both goals would be to find one molecule or pill that would act as both potentiator and corrector. "We have a very aggressive drug discovery pipeline to look for correctors and potentiators," Beall says.

He points to two early-stage programs—one focused on structure-based drug design and the other on combining existing drugs—that are particularly interesting and could quickly produce results.

In 2006, the CF Foundation enlisted Epix Pharmaceuticals to pin down CFTR's actual structure, which could help scientists design better drugs and understand how drugs in the clinic interact with it. With nearly 1,500 amino acids and five domains, "this is a very large protein," says Sharon Shacham, Epix' senior vice president of drug development. Crystal structures of some of the intracellular parts of the protein existed, but a complete picture of the broken channel has eluded the CF community.

Starting with the existing structural information, Epix created a model of CFTR, using homology modeling to generate a picture of the unknown intracellular components and in-house technology to predict the extracellular domain. The company then put the model through several tiers of validation, including comparing it with known biology and looking at its energy profile, a good test because the chloride channel is so negatively charged.

In January, the CF Foundation approved the structure, triggering a milestone payment to Epix and launching the next part of the collaboration: screening for small molecules that will lock into the binding pockets identified in the model. Epix started with a crude screen of its virtual library of about 4 million small molecules. It then chose 150,000 to 200,000 of these compounds and looked at how they dock into the binding pocket of ΔF508, the most common CFTR mutation. That pool was winnowed down to about 200 contenders, which then moved out of the virtual world and into a physical lab for biological testing.

Shacham says the company has already found a scaffold that has both corrector and potentiator properties, and it is moving forward to optimize lead compounds. The CF Foundation is encouraged by the results so far. "The Epix program is very sophisticated, and we've gotten some pretty interesting compounds that are showing a good biological effect," Beall says.

Meanwhile, the biotech firm CombinatoRx is screening a large field of approved and investigational drugs to see whether combining known treatments would have any effect on CF. Diseases inevitably progress along multiple pathways, and perturbing only one is usually not the most potent method of interrupting that course, says Jane Staunton, senior director for therapeutic research at CombinatoRx. Even with a disease such as CF that boils down to a single genetic mutation, "there's still a complexity in the biology that allows combinations to be really powerful," she adds.

CombinatoRx's library of 3,000 compounds includes investigational drugs and the roughly 2,000 approved drugs in existence. Filing through all the drug combinations would be a daunting task, so the company has developed ways of prioritizing its screening efforts. The company has conducted the initial screen and is evaluating data, a process made easier because it is using many known compounds for which a wealth of in vitro information already exists. The next step is to put the most promising molecule combinations through a suite of secondary assays.

"The goal is to get something into the clinic as soon as we can," Staunton says. The ideal outcome, she says, would be to find a combination therapy that would repair multiple defects.

Moving Through The PIPELINE Small-molecule drugs have the potential to treat the underlying genetic defect that causes cystic fibrosis
View Enlarged Image

Although new therapies hold promise for existing CF patients—particularly if damaged chloride channels can be repaired or if activating alternative channels draws more liquid into the lungs—the best results will likely be seen in those who can start taking new drugs at as young an age as possible. For example, Vertex' drugs have the potential to improve liver and intestinal function in patients young and old, but older patients with irreversible damage to their pancreas would have to continue taking enzymes.

Doctors say a new wave of research into CF drug delivery, however, could benefit everyone. That hour or more that patients spend twice daily on the nebulizer is a lot to ask, UNC's Boucher says. He believes the next breakthroughs will come from combining agents, both new and old, into a single-administration mist.

Boucher is a general pulmonologist, so in addition to his CF patients he sees many asthma sufferers who take a few puffs on an inhaler in the morning and evening. He doesn't see why that type of approach couldn't work for CF. "If we could reduce all the CF drugs to dry powders that could be puffed, that would be great," he says.

Designing better delivery systems for drugs is critical, Beall agrees. Patients would have an easier time maintaining their routine if it were less time-consuming and their medicines were more transportable. "That's going to be the next revolutionary movement," he says. If every goal is met, scientists are certain this will become a disease that patients live with rather than die from.

As for Youngblood, she managed to sleep a little later during summer vacation, but recently, she left home to start her freshman year at Macalester College, in St. Paul, Minn. Many adults with CF struggle to balance their therapy with work and social life, and her doctor has already talked to her about sticking to the schedule while she's away. The speech may not have been necessary, as Youngblood can attest: "I just feel better when I do it."

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ISSN 0009-2347
Copyright © 2009 American Chemical Society

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