Blood Relatives.

First-generation artificial blood is about to hit the market

Last year, the tally of blood transfusions climbed to a record high. More people are donating blood than ever before, but a rapidly aging society is using it up even faster as the number of elective surgeries and medical treatments requiring blood transfusions continues to rise.

This year, things are starting out even worse. "We have in fact seen the worst ... in memory, in terms of blood availability," says Harvey G. Klein, president of the American Association of Blood Banks in Bethesda, Md.

In 1999, the U.S. Food and Drug Administration put a ban on blood donations from people who lived in the United Kingdom for more than 6 months between 1980 and 1996. Of concern was the spread of the human version of mad cow disease, Cruetzfeld-Jakob disease, that had hit the United Kingdom. More recently, an FDA advisory committee recommended adding people from France, Ireland, and Portugal to the ban. The spread of AIDS and other bloodborne diseases such as hepatitis has also diminished the blood supply by excluding potential donors.

If only there were substitutes that could fill some of blood's roles in the body, artificial substances that would be free of the supply constraints and contamination vulnerabilities of the real stuff. Besides the roles these substitutes could play in general surgery, such products could save lives during emergencies and major disasters in which blood isn't readily available. It could also be a medical boon to developing countries that don't bank blood.

For decades, researchers have sought to develop a partial replacement for blood. Now, several companies are about to release the first line of artificial blood products. "I've watched this field over the last 20 years, and this is the most promising that it's been," says George Nemo, head of the transfusion-medicine program at the National Heart, Lung, and Blood Institute in Bethesda, Md.

Half the blood supply could potentially be replaced with the new substitutes, claims Robert M. Winslow, president of Sangart, a San Diego-based company developing one of them. Although these products are far from perfect--and researchers are already developing a more sophisticated generation of artificial blood--they carry the oxygen that will keep people alive.

Blood is a wondrous concoction of red blood cells, white blood cells, platelets, and plasma--which is itself a cocktail of proteins, carbohydrates, hormones, and other biochemicals--that together fight infections, heal wounds, deliver oxygen, and remove wastes.

The molecular heart of blood's oxygen-carrying ability is the protein known as hemoglobin, which jam packs red blood cells. More than 250 million hemoglobin molecules can crowd inside a single cell. Each hemoglobin scoops up to four oxygen molecules from the lungs and carries them to all of the body's other tissues. Once depleted of its oxygen cargo, a hemoglobin molecule snatches up carbon dioxide made by cells and brings the gas to the lungs, where it's exhaled.

Hemoglobin has been a favorite starting point for developers of blood substitutes. For one thing, different blood types, such as type A and type B, are based on different sets of proteins, known as antigens, that cover the surfaces of red blood cells. If a doctor gives a patient the wrong type of blood, that person's immune system will reject the foreign cells.

In the 1960s, researchers tried to circumvent complications from cell-surface antigens by making a blood substitute using free hemoglobin extracted from cells. Surprisingly, the naked molecule turned out to be toxic. Normally, hemoglobin exists as a molecule of four tightly bound units. But outside the red blood cell, the units fall apart. In animal studies, the hemoglobin fragments caused kidney damage.

In the 1970s, the Department of Defense continued supporting the quest to develop free hemoglobin into a blood substitute for wounded troops. This work led to ways of chemically rejoining fragmented hemoglobin molecules. Around the same time, other researchers were stabilizing whole hemoglobin molecules by polymerizing several of them into sturdier complexes.

A turning point for blood-substitute efforts came in the early 1980s with the rise of AIDS and the realization that HIV could spread the disease through blood. These fears built into "hysteria about the blood supply," recalls Steven Gould, president of Northfield Laboratories in Evanston, Ill. The threat of tainted blood thrust the quiet field of blood-substitute research into the limelight, he says.

The demand for a safe blood product drove companies to try to quickly bring apparent laboratory successes with hemoglobin-based substitutes into clinical trials. "We were going for the home run" without fully understanding all the effects hemoglobin could have, says Nemo.

Baxter Healthcare of Deerfield, Ill., entered the fray with a product called HemAssist, which is based on a hemoglobin molecule with enhanced stability. The company suffered a setback, however, during clinical testing of HemAssist in trauma patients. Of 52 patients receiving the blood substitute, 24 (46 percent) died. Of 46 patients getting a saline solution, only 8 (17 percent) died. The company-sponsored researchers reported the results in the Nov. 17, 1999 JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. Baxter halted the research.

"We get surprised by [hemoglobin] all the time," says Abdu Alayash, a product reviewer at FDA. "This is an extremely reactive molecule that can interact with a number of biological molecules in the body."

Researchers working with clumps of polymerized hemoglobin molecules have had more success than those working with single-but-modified hemoglobin molecules have. The most likely reason for this, says biochemist John Olson from Rice University in Houston, is that the molecular clumps are so much bigger than single molecules, which are small enough to slip out of blood vessels. Once out, the molecules gobble up nitric oxide in the vessels' smooth muscle tissue. The resulting drop in nitric oxide prevents those vessels from relaxing, a condition that leads to high blood pressure. As it turns out, polymerized hemoglobin is too big to slip through blood vessels and take up nitric oxide, so it's less prone to raise blood pressure than lone hemoglobin molecules are, Olson says.

Researchers have been taking all of these lessons to heart. Several companies are nearing the finish line in the competition to be the first to put a polymerized-hemoglobin blood substitute on the market. Their sources of hemoglobin include cows' blood and unused banked blood older than its 42-day shelf life.

Biopure Corporation of Cambridge, Mass., is a frontrunner in the race with a product based on hemoglobin extracted from the red blood cells of cows. The company has completed clinical trials of the product, called Hemopure, in the United States, Europe, Canada, and South Africa. It's already filed for approval to sell Hemopure in South Africa and will soon do the same in the United States and Europe, says Maria Gawryl, vice president of research and development at Biopure.

Close behind is Northfield Laboratories with its polymerized product, PolyHeme, which is based on hemoglobin extracted from outdated human blood. PolyHeme is in the third phase of clinical trials, the final leg of testing before the company can file for approval from FDA.

Hemosol in Toronto is in a similar position, with a polymerized hemoglobin product, called Hemolink, made from outdated human blood. The company has completed the final phase of clinical trials in Canada and has ongoing clinical trials in the United States.

These products tend to be costly and outdated human hemoglobin is often limited in supply. So another company has taken an approach that doesn't rely on hemoglobin at all.

Alliance Pharmaceutical in San Diego has created a blood substitute made of synthetic chemicals called perfluorocarbons. These chains of carbon molecules with many attached fluorine atoms can carry large amounts of oxygen and carbon dioxide.

"What [such a product] has going for it is that it's cheap and can be produced in large amounts," says Winslow of Sangart. Perfluorocarbons may also provide an alternative for patients who decline any natural blood products for religious reasons.

But Alliance recently suffered a setback. During a clinical trial, patients receiving the perfluorocarbons product--called Oxygent--during cardiac surgery experienced a higher rate of stroke than patients in a control group. The company suspended the trial in January to investigate the cause of the strokes.

"We unfortunately have to take a little time out here in our clinical development before we forge ahead, but this is a huge market that's not going away," says Peter Keipert, director of Oxygent development at Alliance.

Other perhaps more promising products are in the works but further back in the developmental pipeline. For example, to reduce hemoglobin's tendency to pick up nitric oxide and raise blood pressure, Rice University's Olson aims to alter the molecule's structure and properties.

As a start, he's studying myoglobin, which normally provides muscle cells with oxygen, as a model for hemoglobin. He and his colleagues have engineered myoglobin molecules that remove much less nitric oxide than normal.

Both myoglobin and hemoglobin are proteins with pockets that catch oxygen molecules. To discourage myoglobin from catching nitric oxide as well, the Rice researchers replaced the small amino acids leucine and valine in the protein's oxygen pocket with tryptophan, a larger amino acid. The substitution reduced the opening, allowing the altered myoglobin to carry oxygen but not nitric oxide. Unfortunately, the modified myoglobin also locked onto the oxygen too tightly. Olson solved that problem by making another amino acid substitution that weakened myoglobin's hold on the oxygen. Animals given this fully modified myoglobin have maintained normal blood pressure.

Baxter Healthcare has worked with Olson's group and made similar changes in hemoglobin. Animals given these modified hemoglobin products have also maintained normal blood pressure.

Researchers at Sangart are trying to increase the length of time hemoglobin products last once infused into the bloodstream. At the moment, these only last a few days. Now, the researchers are attaching large soluble chains of polyethylene glycol to individual hemoglobin molecules. These chains seem to keep the hemoglobin circulating longer before they're filtered out of the body, says Winslow. The polymers also envelop the hemoglobin molecule in a layer of water, creating a complex too large to leak out of the blood vessels, he says.

Taking another tack, researchers at SynZyme in Irvine, Calif., are trying to add more functions of whole red blood cells to hemoglobin products. Red blood cells, for instance, contain enzymes that rid the body of harmful oxygen radicals. These destructive molecules proliferate when tissues lose blood. In an effort to create a product that mimics the protective function of red blood cells, the researchers are attaching antioxidant enzymes, such as catalase and superoxide dismutase, to hemoglobin.

Thomas Chang of McGill University in Montreal is doing something along those lines. However, instead of attaching enzymes to hemoglobin, he and his colleagues are designing a biodegradable membrane that will contain hemoglobin, antioxidant enzymes, and other components of a red blood cell. Unlike real red blood cells, however, these artificial cells would be antigenfree.

Genetic engineers also have joined the quest for blood substitutes. Using recombinant DNA technology, researchers are altering the genes that encode hemoglobin and then putting those genes into Escherichia coli bacteria, which then produce modified molecules. Chien Ho and his colleagues at Carnegie Mellon University in Pittsburgh have made altered hemoglobin that is stable outside a red blood cell. They reported their research in the Nov. 14, 2000 BIOCHEMISTRY.

None of these products will duplicate all the functions of whole blood. Red blood cells have a membrane that allows them to circulate in the body for a couple of months, whereas the kidneys filter out blood substitutes in days. "You can't keep people in the hospital and keep pouring this stuff through," notes Rebecca Haley, chief medical officer of biomedical services of the American Red Cross in Washington, D.C.

The products coming to market will serve the emergency needs of trauma victims and patients undergoing surgery, says Klein. However, at least 30 percent of whole blood goes to people with diseases requiring longer-term blood replenishment, he points out. Bone marrow-transplant patients, for example, need blood or a substitute until their bodies replenish their own supply of red blood cells, which can take weeks.

As a consequence, says Haley, for the foreseeable future, people will still need to donate the real thing, even as artificial blood substitutes finally begin finding their medical niches.