Issue Date: February 23, 2009
IT'S BEEN TWO DECADES since academic and industrial research of blood substitutes took off, yet the field has never been as turbulent as it is now. A diverse cast of characters play leading roles in this tumultuous field, including doctors, military scientists, Food & Drug Administration regulators, academics, and a handful of companies trying to develop products that can deliver oxygen within the body. Some argue that blood substitutes are necessary and safe. Others say blood substitutes are dangerous and clinical trials should be abandoned.
R&D has focused primarily on mimicking the ability of red blood cells to bind oxygen. The most common approach has been to take hemoglobin—the key oxygen-binding protein in blood—from various sources and make it work outside of red blood cells. A major challenge is the potential toxicity of hemoglobin when it is outside a red blood cell, which can be deadly.
Ethical questions involving patient enrollment in clinical trials, as well as conflicts of interest by prominent participants in the debate, have also hurt the field. Private and public funding is becoming scarcer, and a few companies have cut staff, leaving some wondering whether artificial blood research and development has suffered a mortal blow.
The drive to develop blood substitutes has come from a variety of places. Even after blood banks emerged in the 1930s, the pesky issue of blood-group incompatibilities between donors and recipients spurred academics to seek an alternative supply of blood. Then there are those, such as Jehovah's Witnesses, who object to using donated blood on religious grounds but might be open to synthetic substitutes. The military has also been an early and steadfast supporter of research to develop a blood substitute. Donor blood has only a 28-day shelf life and requires cold storage. On the front lines of war, it's therefore difficult to find a steady supply of transfusable blood to treat casualties.
"And then HIV came," says Thomas Chang, an emeritus professor at McGill University, in Montreal, who got involved in blood-substitute research in the late 1950s. Fears of an unsafe blood supply because of potential donors with HIV and other infections prompted funding agencies, the military, and venture capitalists to pour billions of dollars into the development of blood substitutes.
The field thus experienced rapid growth in the '80s and '90s that was further bolstered by the emergence of severe acute respiratory syndrome (SARS) and West Nile virus. More than 50,000 blood-substitute patents have now been filed.
Yet the quest for a blood substitute has rather rudimentary beginnings. Before the advent of blood banks, doctors faced with mortally bleeding patients tried everything from salt water to wine as a blood proxy. Legend has it that urine was even infused into patients.
Nowadays, those wanting to make blood mimics have sought more scientifically sound inspiration for their creations. Although blood is a complex mixture of immune cells, hormones, platelets, and proteins, red blood cells arguably play the most vital role because they ensure the delivery of oxygen throughout the body. As such, the blood-substitute field primarily aims to mimic the red blood cell's ability to bind oxygen in the lungs and release it elsewhere.
The first serious attempts to make a blood substitute came in the early part of the 20th century, when medical researchers realized that red blood cells are packed with the protein hemoglobin. Hemoglobin consists of four subunits, each containing a heme group that clutches oxygen as the red blood cells travel through the vasculature. Early on, doctors took human and animal blood, extracted hemoglobin from its red blood cell packaging, and injected the protein directly into patients.
Those experiments failed because outside the confines of a red blood cell's protective capsule, hemoglobin breaks into two pieces, overwhelming the kidney. Some patients died.
When researchers realized dissociation was a problem, they began to chemically stabilize hemoglobin to prevent it from falling apart. Most oxygen carriers in development today still employ the same strategy.
For example, Cambridge, Mass.-based biotech firm Biopure links neighboring hemoglobins together through molecules of glutaraldehyde, says W. Richard Light, vice president of technology development at Biopure. The glutaraldehyde binds to lysine side chains located on the surface of neighboring proteins.
Mixtures of two to eight hemoglobins connected in this way make up Biopure's Hemopure product. It received clinical approval in South Africa in 2001 for use as an oxygen carrier during surgeries on anemic patients, and it is the only hemoglobin-based blood substitute permitted for use in humans. The company has not gotten FDA approval for use in people in the U.S., although the agency permits use of a similar product in dogs.
The source for Biopure's hemoglobin is cow blood. Other companies, such as San Diego-based biopharmaceutical firm Sangart, use human hemoglobin from outdated blood bank donations.
Instead of connecting several hemoglobin molecules together as Biopure does, Sangart attaches up to eight polyethylene glycol (PEG) molecules onto a single hemoglobin. The PEG keeps hemoglobin together and in circulation, says Howard Levy, Sangart's chief scientific officer.
Keeping hemoglobin-based oxygen carriers in circulation turns out to be a common challenge. Although such products survive on a shelf for months or years, once injected into humans they are in circulation for only up to 48 hours, compared with 120 days for new red blood cells. This means that in clinical situations, readministration of the product or a top off with donor blood would be necessary.
THE HEMOGLOBIN protein itself presents a more serious problem. Although its power to bind and transport oxygen to the tissues makes it an attractive molecule to develop as an oxygen carrier, outside the confines of a red blood cell's membrane, hemoglobin can cause harm.
Hemoglobin doesn't just bind oxygen and carbon dioxide. It also has a penchant for nitric oxide (NO), a gas that plays a role in keeping blood vessels taut or open. Hemoglobin can remove NO from blood vessels by converting it to nitrate (NO3–), explains John S. Olson, a chemist at Rice University.
Depleting NO can cause blood vessels to contract, which makes it more difficult for blood to flow and raises blood pressure. Many academics and regulators say NO scavenging is probably one of the causes of heart attacks and deaths observed during clinical trials of blood substitutes. Some companies have tried to make their hemoglobin-based blood substitutes larger through polymerization or other chemical modifications to thwart the protein from accessing the nooks and crannies of a blood vessel, where NO lies, Olson says.
There's another concern about sending hemoglobin products through the vasculature without the red blood cell packaging: "The iron in the hemoglobin can start to spontaneously oxidize, creating free radicals that can ultimately cause cytotoxicity in tissue and organs," explains Abdu I. Alayash, who heads the Laboratory of Biochemistry & Vascular Biology in FDA's Office of Blood Research & Review. The oxidized hemoglobin can also be self-destructive, leading to protein damage and increased toxicity, explains Chris Cooper, a biochemist at the University of Essex, in England.
Some of these problems drew scrutiny in 1998, when Baxter International became the first company to go into clinical trials with a hemoglobin-based oxygen carrier. According to the Wall Street Journal, the company halted the study when nearly half of the patients receiving the hemoglobin-based carrier died, compared with 17% in the control group. The company later pulled out of the field altogether.
Deadly clinical trials haven't been the only problem. Other clinical trials have raised the alarm among bioethicists and patient rights groups because of the manner in which patients were enlisted. For example, Evanston, Ill.-based Northfield Laboratories gained FDA approval to run a human clinical trial of its polymerized human hemoglobin, PolyHeme, in trauma patients. Concern arose because the company enlisted unconscious trauma patients in the trial without their consent; it did so legally, by means of an FDA exemption from requiring informed consent from patients participating in clinical trials for some emergency medicines. After critical media reports, several hospitals pulled out of the trial.
In 2007, Northfield published results for a clinical trial of 712 patients who were severely hemorrhaging. Forty-two individuals receiving the blood substitute died, compared with 35 in the control group receiving saline solution or donated blood. Despite these results, Northfield has since filed a request for FDA to approve PolyHeme for "life-threatening red blood cell loss when an oxygen-carrying fluid is required and red blood cells are not available," according to the company's website. Northfield, which declined to be interviewed for this article, expects an answer from FDA this spring.
AMID GROWING controversy about hemoglobin-based oxygen carriers, many say the biggest blow to the field came last spring.
FDA had invited stakeholders from industry, regulatory agencies, academia, medicine, and the military to discuss hemoglobin-based oxygen carriers at a weekend workshop in late April. One reason for the workshop was that "there have been no successful clinical trials to date," from surgery to trauma indications, says Toby Silverman, a senior medical adviser in FDA's Office of Blood Research & Review. There was "a growing recognition that we needed to talk with experts about why the field was having so much difficulty with clinical trials, what was the nature of the adverse effects, and what was the state of the art in terms of preclinical work, basic biology, and basic understanding of these adverse events," she notes.
The Friday before the weekend workshop, a National Institutes of Health medical researcher named Charles Natanson, in collaboration with Public Citizen, a consumer advocacy organization, published a critical metastudy of hemoglobin-based oxygen carriers in the Journal of the American Medical Association (2008, 299, 2324). It reported that individuals involved in clinical trials of these compounds had a 30% increased risk of death and nearly triple the risk of a heart attack. An associated editorial by Dean A. Fergusson, from the Ottawa Health Research Institute, in Canada, called for a halt of clinical trials in humans. "We need to understand the mechanism of action of the hemoglobin-based oxygen carriers a lot better before they are tested in humans," Fergusson tells C&EN.
The publication of the JAMA article just before the workshop "made for a very tense meeting," says the University of Essex' Cooper, who attended. "On one side were the industry people, and on the other was Natanson and his supporters. The Navy scientists, in full uniform, were in the middle, trying to keep people focused on how to deal with toxicity issues and on moving the field forward."
Since the publication of the JAMA study, several companies have posted responses on their websites attacking the statistical veracity of the analysis and the assumption that hemoglobin-based oxygen carriers can be grouped together as a class.
"All these molecules contain hemoglobin, but that's where the similarity ends," Biopure's Light says. The different products have different sizes, oxygen binding, transport properties, and methods of purification, he adds.
Others disagree. "All of these products start with some form of molecular hemoglobin, most are more than 90% molecular hemoglobin by weight, and all of the reported toxicities are associated with the products' hemoglobin component," wrote John R. Hess of the University of Maryland School of Medicine in a Transfusion editorial (2008, 48, 2051).
"We have to look at the totality of evidence," Fergusson says. "The products are all meant to do the same thing, so I think it is fair to call them a class of interventions."
Like Light, Jonathan Jahr, a medical doctor at the University of California, Los Angeles, who owns Biopure stock and has run clinical trials for the company, says he doesn't think it is fair to lump hemoglobin oxygen carriers together as a group. But he notes, "I do feel the JAMA article authors pointed out some issues that warrant repeating: Most companies have been reluctant, and still are, to release their products to independent investigators for study, and some are to be criticized for not publishing all the data."
JAMA has also published several letters to the editor from doctors citing the need for oxygen-based carriers in developing nations or remote regions where blood banks are understocked or contaminated, or for compassionate-use cases such as those involving Jehovah's Witnesses. All letters included disclosures of at least one writer's financial connections to the hemoglobin-based oxygen carrier industry.
THEN ANOTHER twist emerged. Natanson, the JAMA author, had failed to report a conflict of interest, namely a blood-substitute patent that aimed to counteract some of the side effects Natanson criticized in the existing hemoglobin-based carriers. Natanson's name was later removed from the patent. However, last fall, Biopure's lawyers launched a defamation suit against Natanson, noting the conflict of interest and the fact that Natanson had sent the JAMA article to medical authorities around South Africa to alert them of the metastudy's results. According to the October 2008 South African Medical Journal, health authorities in at least one district of the country instructed doctors to stop using Biopure's product. Natanson declined to be interviewed for this article.
The metastudy, coupled with the current tough economic situation, has been a "death blow" to the blood-substitute research community, Light says. The products are "not toxic, but public opinion has been swayed by inflammatory comments with no merit," he says. Biopure has downsized by 90%, leaving fewer than 10 employees. Last December, FDA denied a request from Navy scientists to start a clinical trial with Biopure's product for the resuscitation of casualties who had lost a severe amount of blood in locations where no blood transfusions were available.
Other companies also face challenges. Sangart is currently looking for new medical indications for its product Hemospan after a Phase III clinical trial in orthopedic surgery patients, completed last year, "was unable to demonstrate clinical benefit in terms of improved outcomes," the company's website notes. Sangart has chosen not to pursue trauma applications for Hemospan in favor of using the product to top off oxygen in tissues that become oxygen-depleted because of surgery or ailments such as stroke, Levy says. Although Sangart cut quality assurance staff last year, Levy says it plans to hire marketing staff this year, adding that he remains "optimistic" about the future.
Indeed, small blood-substitute biotech companies continue to establish themselves. For example, Dallas-based HemoBioTech is seeking FDA approval to begin clinical trials for its modified hemoglobin product, which uses adenosine as a hemoglobin linker.
Yet in academic circles, the negative publicity has made it more challenging to get funding, Rice University's Olson says. He continues to work on newer generation products that aim to avoid the oxidative side effects of cell-free hemoglobin.
For example, Olson has used protein engineering to block the pocket in hemoglobin where NO is captured before being oxidized to try to avoid NO scavenging. He is currently working on ways to scale up bacterial production of the modified hemoglobin so that it can be made at a high enough volume to be industrially useful.
Others are trying to envelop hemoglobin and oxidation-protective enzymes in an artificial cell made of PEG and polylactic acid copolymers. Some are using stem cells to build red blood cells, which could avoid the problems of cell-free hemoglobin altogether. Researchers like Cooper are investigating the redox reactions of hemoglobin to better understand oxidative damage.
"I don't think we should extinguish research in the area," Fergusson says. "But we need to know much more, and we need to make sure we do things right before going into humans again."
FDA's Silverman says that the workshop and JAMA paper "did bring to light the need for very careful decision-making about clinical trials of blood substitutes, particularly because there have been observed toxicities for all of the products, and there's been a lack of benefit in previous human trials."
Despite the controversy, FDA is still planning to allow human testing of the products, Silverman says. "The bar might be higher, but FDA will continue to allow clinical trials in consented studies when risks are deemed to be reasonable." But for studies that require a consent waiver, trials will only be permitted "where there is a likely benefit," she says.
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