Searching for Medicine's Sweet Spot

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Science  23 Mar 2001:
Vol. 291, Issue 5512, pp. 2338-2343
DOI: 10.1126/science.291.5512.2338

Long avoided by chemists and biologists, sugar-based drugs are suddenly on medicine's menu and garnering impressive early reviews

Just over a decade ago, a bug called Haemophilus influenzae type b (Hib) wrecked the lives of some 25,000 children a year in the United States alone. Rather than causing the flu, as its name suggests, Hib produced bacterial meningitis in 60% of infected children, 10% of whom died. Those who survived often suffered permanent damage, ranging from mild hearing loss to mental retardation. But thanks to a sugar-based vaccine, Hib has been virtually eliminated from the United States, most European countries, and a growing number of developing nations. The World Health Organization is now pushing the vaccine's use to prevent the estimated 400,000 annual Hib-related deaths worldwide.

The Hib vaccine isn't the only sweet success for sugar-based therapies. The anticoagulant heparin—a complex sugar, or carbohydrate—has long been the number-one-selling drug in the world. Top-selling protein drugs, such as the red blood cell booster erythropoietin (EPO), bristle with sugars that ensure that these molecules stay in circulation long enough to carry out their task. And two new flu drugs approved for use in 1999 work by attacking a sugar-busting enzyme that influenza viruses use to help them exit infected cells.

But these triumphs have been hard won. Six decades elapsed between the discovery of the sugar groups on Hib that induce an immune response and the development of an effective vaccine. Amgen, the maker of EPO, regularly throws out as much as 80% of its protein because the attached sugars are incorrect. Meanwhile, the 1990s saw a series of biotech companies go belly up after their carbohydrate drugs failed in clinical trials (see p. 2340). “The field has struggled, there's no denying it,” says David Zopf, vice president of Neose Technologies in Horsham, Pennsylvania.

This struggle has long confined research on carbohydrates to a small niche in biology. The field didn't even have a proper name until 1988, when Oxford University biochemist Raymond Dwek coined the word glycobiology during an appearance on a British morning television show. “In a world focused on nucleic acids and proteins, there really wasn't that much interest in carbohydrates except from people studying energy metabolism and the few of us who were toiling away under the radar,” says Dwek, who is now director of Oxford University's Glycobiology Institute in the U.K. John Magnani, president of GlycoTech, a carbohydrate- focused biotech firm in Rockville, Maryland, says that even today, carbohydrate research is “the Rodney Dangerfield of pharmaceutical research. It gets no respect.”

But recent advances in the study of carbohydrate chemistry and biology are beginning to turn the tide. Chemists have begun to crack a long-standing problem: producing carbohydrates in quantities large enough to study in biological systems. Like proteins and nucleic acids, carbohydrates are polymers of relatively simple molecules linked together. But unlike the amino acids in proteins and the nucleotides in nucleic acids that link up like boxcars in a train, sugar chains can branch and twist, giving them a multitude of three-dimensional shapes. That structural complexity makes carbohydrates difficult to analyze and extremely hard to make. But new automated methods of synthesizing large quantities of carbohydrates promise a dramatic change (Science, 2 February, p. 805); similar developments spurred the study of proteins and nucleic acids in the 1970s and 1980s.

Two decades of genetic and biochemical studies by researchers who toiled largely in obscurity have also spelled out many critical roles played by carbohydrates and identified potential drug targets. Sugars are ubiquitous in cells. They dangle from nearly all the protein and many of the fat molecules in the body. These combination molecules, called glycoproteins and glycolipids, dot the outer surfaces of all cells and serve as cellular identification tags to the surrounding world. The body uses them to say something as general as “I'm a human tissue, I belong here,” or as specific as “I'm injured, send help from the immune system over here.” Cancer cells use sugar groups on their surfaces to slip past the immune cells looking to do them in as they migrate through the body. Pathogens rely on the glycoproteins and glycolipids on cell surfaces to home in on their tissue of choice in their favorite host species and to spread themselves from cell to cell. Harmful inflammatory reactions are often triggered by carbohydrates, as is blood clotting.

“Carbohydrates are central to many processes that are at the core of important diseases, and now that we understand some of those roles, it's not surprising that this has become a hot topic at drug companies,” says Christian Raetz, a glycobiologist who helped Merck get into the field before he returned to academia as chair of the biochemistry department at Duke University Medical Center in Durham, North Carolina.

Now, dozens of new carbo-related compounds are in clinical trials, aimed at treating conditions ranging from inflammation and tissue rejection to hepatitis and cancer. “The result is that glycobiology's future now looks pretty bright,” says Zopf. “Glycobiology has finally become part of the mainstream,” adds Hudson Freeze, a longtime glycobiology researcher and director of the glycobiology program at the Burnham Institute in La Jolla, California. “We're no longer a boutique science.”

Interrupting inflammation

One of the first critical roles played by carbohydrates began to come into focus in the late 1980s. Several groups independently cloned the genes for three human carbohydrate- binding proteins that play a role in attracting leukocytes, or white blood cells, to injured sites in the body. Called selectins, these proteins appear on the surface of the endothelial cells lining blood vessels after injured tissues nearby release powerful signaling compounds known as cytokines. The availability of the cloned genes led to a flood of publications in the early 1990s showing that selectins bind to a distinct carbohydrate structure, called sialyl Lewis × (sLex), on the surface of circulating leukocytes. This binding acts as a kind of brake that slows down leukocytes circulating in the bloodstream, causing them to roll along the injured blood vessel wall and allowing them to slip into the injured tissue.

Hold it.

Selectin proteins and their sugar-based targets slow the passage of white blood cells in circulation, allowing them to enter damaged tissue. Interrupting this binding may prevent inflammation.


Although those leukocytes launch the healing process, they can also lead to inflammation, which itself can damage tissues. That prompted several groups to try to prevent inflammation by blocking the ability of selectins to bind to their sLex targets. Early experiments by Ajit Varki and his colleagues at the University of California, San Diego (UCSD), offered initial hope. Their work in mice showed that blocking a selectin subgroup called L selectin from binding to sLex reduced inflammatory responses in damaged blood vessels.

Since then, the track record for getting selectin inhibitors to work in humans has been spotty at best. In 1995, researchers at Boulder, Colorado-based NeXstar Pharmaceuticals, now a part of Gilead Sciences in Foster City, California, developed potent selectin inhibitors in collaboration with Varki's group. But when the company's inhibitors failed to work in animal disease models, the project died on the vine. Cytel, formerly of San Diego, made it farther. Its compound, intended to prevent damage to tissues after blood starts flowing again after a heart attack, stroke, or tissue transplantation—a condition known as ischemia reperfusion injury—made it through human safety studies. But final-stage clinical data showed no benefit from the drug, and the company closed shop. “Historically, this has been a tough area to be in,” says Gray Shaw, who heads drug development for a selectin inhibitor at Wyeth, the pharmaceutical arm of American Home Products based in St. Davids, Pennsylvania.

Wyeth is betting it can do better with a compound called PSGL-1 that binds to another selectin subtype called P selectin. P selectin is expressed not only on endothelial cells but also on platelet cells, causing these red blood cells to stick to leukocytes and create blood clots. “By inhibiting P selectin, we hope to not only prevent ischemia reperfusion injury but also the subsequent clotting events that can reocclude a vessel that's just been opened,” says Shaw. Wyeth is currently conducting phase II trials with a soluble recombinant form of PSGL-1, and Varki, for one, is optimistic. “I think that the Wyeth drug is the first really good selectin inhibitor that has been given a chance to prove itself,” he says.

Inhibiting P selectin may also prove to be useful for stopping the spread of tumors. Metastasizing tumor cells grab onto P selectin on platelets and use them as a protective shield against immune system cells. In the 13 March issue of The Proceedings of the National Academy of Sciences, Varki and his UCSD colleagues reported that heparin treatment, which has been used with mixed success as part of chemotherapy, dramatically slows tumor metastasis by binding to P selectin on platelets before cancer cells can do the same. “This appears to markedly reduce the long-term organ colonization by tumor cells,” says Varki. The response is inconsistent, however, perhaps because the chemical makeup of heparin is variable, and the clotting problems that can result from heparin therapy make it unlikely that heparin will be widely used as a chemotherapy agent. Other P selectin inhibitors may fare better, however.

Cancer vaccines

Stopping tumor cells from binding selectins isn't the only way researchers hope to use carbohydrates to block cancer. Transformed tumor cells hide from normal immune surveillance by displaying glycoproteins and glycolipids on their surfaces. Some researchers are now trying to turn the tables by using the carbohydrate chains from these glycomolecules as vaccines. “The idea is to manipulate the antigens in such a way that they become visible to the immune system,” says Alan Houghton, chief of clinical immunology at Memorial Sloan-Kettering Cancer Center in New York City. “We and others have been able to do that, and the immune system will then make a concerted, and apparently successful, attack on tumors.”

Several companies and universities are conducting clinical trials of carbohydrate-based anticancer vaccines. Houghton and his colleague Philip O. Livingston, for example, have been heading a team that is using synthetic carbohydrate antigens prepared by chemist Samuel Danishefsky and members of his laboratory at Sloan-Kettering and Columbia University.

While Danishefsky's team worked out how to synthesize carbohydrate cancer antigens with names such as Globo-H, which is associated with breast cancer, and Fucosyl GM1, which is isolated from small cell lung cancer, Livingston was figuring out how to boost their otherwise lousy ability to trigger an immune response. The solution was to link these carbohydrates to keyhole limpet hemocyanin, a strongly immunogenic protein isolated from a marine mollusk, and then deliver the twosome with another immune booster. The Sloan-Kettering group is now conducting phase II and phase III trials in late-stage patients for whom more conventional therapies have failed. Patients receive immunizations once weekly for a month and then every 3 months for the duration of the trial. Results are expected within the next year.

In the meantime, Danishefsky's group is pressing ahead on a next-generation vaccine: a chemically linked combination of several individual carbohydrates known as “polyvalent antigens.” “Work in the mouse suggests that these polyvalent antigens will do a better job yet,” says Danishefsky, who adds that making the molecules is the most complex synthesis project he has ever undertaken.

Biomira, a biotech firm in Edmonton, Alberta, is also nearing the end of its own cancer vaccine trial. The company's Theratope vaccine uses an antigen known as STn, part of a larger antigen known as mucin-1 found on breast cancer cells. This month, the company completed enrollment in a double-blinded phase III breast cancer trial that will test the vaccine on more than 950 women with metastatic breast cancer. “We'll get our first look at the data in about 6 months and take another look at these patients about 18 months from now,” explains Mairead Kehoe, the company's director of clinical trials. While the waiting game continues, the mood among cancer researchers remains upbeat. “Our expectation is that carbohydrate-based vaccines will stop the metastatic spread of cancer and allow the body to control this disease,” predicts Danishefsky.

Viral deconstruction

Researchers also have big hopes for carbohydrate drugs to stop another kind of invader: viruses. It turns out that even relatively minor interference with the sugars on proteins that make up the viral coats of two major scourges, the hepatitis B and C viruses, can produce big results.

Later this year, United Therapeutics of Silver Spring, Maryland, is expected to begin clinical trials on two antihepatitis drugs discovered as part of a collaboration between Oxford's Dwek and Timothy Block, a viral hepatitis specialist and director of Thomas Jefferson Medical College's Jefferson Center for Biomedical Research in Doylestown, Pennsylvania. Both drugs are variants of natural sugars, and they work by gumming up two glycoprotein-processing enzymes in the endoplasmic reticulum (ER), the site where cells add carbohydrates to newly synthesized proteins.

When the hepatitis B virus invades liver cells, it depends on the ER's machinery to reproduce. But Dwek and Block found that when they added the compound N-nonyl deoxynojirimycin (NN-DNJ) to human liver cells, glycoprocessing was disrupted to a small but crucial extent. The result: The virus couldn't construct its M envelope protein, a critical coat component. These test tube studies show that “inhibition of as little as 6% of cellular glycoprocessing results in a greater than 99% reduction in the secretion of hepatitis B virus,” says Dwek. “There seems to be no effect on the host [cells], but it's a lethal change for the virus.”

Dwek and Block believe these drugs cause viral replication to go awry by preventing the envelope proteins from folding into the correct three-dimensional shape. “We know that there are proteins called chaperonins in the ER that grab onto a new protein's sugars and help the protein fold correctly,” says Dwek. The two scientists suggest that only a small number of misfolded M proteins disrupt the symmetry characteristic of the hepatitis B virus coat and prevent it from escaping the endoplasmic reticulum.

Studies in woodchucks, the preferred animal model for testing potential hepatitis B drugs, confirmed that NN-DNJ stops viral replication cold, with no detrimental effects on the animal's health. “Better yet, we've been unable to find any mutant viruses that can escape this effect,” says Block. “That's one big advantage of targeting a host enzyme and doing so at such a low level.”

That could come as welcome news to hepatitis B patients, who commonly take a drug called lamivudine. The drug has serious side effects, and in 20% of patients the virus develops at least partial resistance within a year, a figure that rises to 53% after 3 years.

Using the same partial interference approach, Dwek and Block recently developed a second sugar compound, N-nonyl deoxygalactojirimycin, that has the same effect on hepatitis C virus in animal tests.

Although this approach has yet to prove itself in humans, a third sugar compound from Dwek's group is well on its way to demonstrating that a subtle adjustment in glycolipid synthesis can benefit at least some patients with Gaucher's disease, one of a class of inherited disorders known as glycolipid storage diseases. Gaucher's disease results from one of many genetic mutations that can either slow or prevent the breakdown of certain glycolipids, which accumulate in storage vesicles and eventually kill cells. Since 1994, Genzyme Therapeutics, headquartered in Cambridge, Massachusetts, has been selling a recombinant form of the enzyme at fault, glucocerebrosidase, under the trade name Cerezyme. The therapy is highly effective, but it requires a 2-hour intravenous infusion as often as three times a week and costs approximately $200,000 a year.

No escape.

Newly approved carbohydrate drugs block the ability of flu viruses to exit infected cells by binding to neuraminidase, a viral protein required for the job. [animation]


Dwek, along with Oxford colleagues Terry Butters and Frances Platt, reasoned that they might be able to restore the body's glycolipid balance—at least in patients whose mutations don't completely destroy the affected enzyme—by decreasing the synthesis of the glycolipids, which also occurs in the ER. This time, they chose a sugar variant called NB-DNJ as their weapon of choice. The results have been promising. “We don't shut down glycolipid synthesis completely, just enough to restore the proper balance between synthesis and degradation,” says Dwek.

Oxford Glycosciences, an Oxford, U.K.-based biotech firm specializing in carbohydrates, has been conducting clinical trials with NB-DNJ, known more prosaically as Vevesca, both alone and in combination with Cerezyme. Last April, the company published initial clinical data in The Lancet demonstrating the drug's safety and hinting at its effectiveness. Last month, the company announced its preliminary analysis of a 6-month phase III study indicating that the compound—which is taken orally—was as effective as Cerezyme at maintaining healthy phospholipid levels. With these promising results in hand, the company is now pressing forward with clinical trials on related compounds for other lipid storage disorders, including Fabry's disease.

This new surge of interest in carbohydrate-based therapies is removing some of the sour taste of the earlier disappointments in the field. Now, glycobiologists are more confident that a spoonful of sugar will not only make the medicine go down, but replace it with something that works better.

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