SEARCHING FOR A SUBSTITUTE
Creating a substitute for human blood has been an elusive dream for many years, but researchers are getting closer than ever before.
Written by Michael Sommermeyer & Julie Toland
Creating a substitute for human blood has been an elusive dream for many years, but researchers at Texas Tech University Health Sciences Center have been able to do what others have not - eliminate hemoglobin's intrinsic toxicity and improve its function as a universal blood substitute.
The World Health Organization reports that 100 million units of blood per year are needed around the world, but currently only 75 million units are available. The United States alone annually uses about 11 million units and a shortage of 4 million units per year is projected by the year 2030. Coupled with the short supply is the fact that blood can only be stored for 42 days. The problem only grows more urgent as nations attempt to cope with the high costs of collecting and testing blood for disease. The World Health Organization reports that of the 75 million units of blood collected around the world, only 20 percent are tested for HIV, hepatitis and a host of other diseases. Most testing occurs in the United States, where the implementation of sensitive screening has reduced the risk of infectious disease transmission to 1 in 60,000 blood transfusions for hepatitis B and 1 in 500,000 for HIV, with intermediate transmission rates for hepatitis C and the human T-cell leukemia virus.
The lack of testing occurs mostly in developing and undeveloped nations. For instance, it is reported that India fails to screen 50 percent of its donated blood for disease, and roughly 10 percent of the country’s paid donors test HIV-positive. Only 16 of the 35 member states of the Pan American Health Organization screen their blood supplies for hepatitis and the AIDS virus. And the World Health Organization is worried about other emerging risks of blood transfusions: retroviruses, prions of human and animal origin, new viruses such as hepatitis G, tick-borne illnesses and a variety of parasites and bacteria.
In a novel research project that has been under way at Texas Tech Health Sciences Center since 1982, scientists hope to replace blood with an effective, safe and inexpensive fluid that has all the essential properties of red blood cells, but without the diseases and short shelf life. Doctors Mario Feola, M.D., professor of surgery; Jan S. Simoni, DVM, Ph.D., research assistant professor of surgery; and the late Peter C. Canizaro, M.D., former professor and chairman of surgery (1982-1990), have numerous patents for their process which allows for the manufacture of a red cell substitute. Scientists at the Eighth International Symposium on Blood Substitutes in November 2000 recognized the process as one of the best likely solutions to the increasing blood shortage problem.
The challenge to create a non-toxic, safe and economical blood substitute has become biotechnology's "holy grail," noted Simoni, a research assistant professor in the Department of Surgery at the health sciences center. He said a universal blood substitute could open significant global markets to a blood substitute that would be pathogen-free and universally compatible with all blood types. Such a product could alter emergency treatment procedures for patients in hemorrhagic shock, prolong the survival time of organs donated for transplantation, improve blood’s oxygen carrying capacity to treat life-threatening illnesses such as heart infarcts and strokes, be used in tumor radio sensitization and be used in the treatment of sickle cell anemia and other hematological disorders.
"There is a $15 billion potential market for the company or research group that develops an oxygen-carrying solution that performs like red blood cells," Simoni said. The U.S. Army recognized the need for a blood substitute following World War II. The loss of blood among soldiers in combat, coupled with the inability to store blood for long periods of time, convinced the military that a solution needed to be found. Traditionally blood has been used when a patient needs an immediate transfusion. However, such transfusions require cross matching of blood types to ensure that a patient does not suffer a severe hemolytic reaction. This step is very impractical on the battlefield. However, oxygen-carrying properties of hemoglobin in solution make it very attractive as the basis for a blood substitute. For many decades, the U.S. Army was the leading force in searching for an effective substitute for blood. Doctors always saw it as a potential lifesaver for anyone needing an immediate transfusion when there is no time to identify blood type.
Since the 1930s, hemoglobin has been experimentally used as a way to increase blood volume and to transport oxygen throughout the body. While it seems like a simple solution, hemoglobin brings with it many problems. The erythrocytic stroma associated with unpurified hemoglobin can be toxic to the body resulting in rapid clotting of the blood, followed by cardiac failure and ultimately asphyxiation. Even stroma free, but not chemically modified hemoglobin, causes damage to the kidneys and other organs. While human hemoglobin in solution is an effective oxygen carrier, it also can fail to release oxygen to the tissues. Release of oxygen from human hemoglobin is a complex chemical reaction regulated by a molecule of 2,3-diphosphoglycerate (2,3-DPG), which allows hemoglobin to function effectively. When the level of 2,3-DPG is upset in any way, which happens outside red blood cells, hemoglobin binds oxygen so tightly that it ceases to function as an effective oxygen provider.
Scientists from around the world have experimented with various approaches to maintain a balance in the chemical makeup of hemoglobin in an effort to maintain its main positive attribute of providing ample oxygen to the body. After much research, scientists found ways to purify hemoglobin and to bind the 2,3-DPG analogues to the hemoglobin molecule that reduced its toxicity and allowed for the release of oxygen throughout the body. Also, the short half-life of hemoglobin in circulation was resolved in various ways. This research created a whole new class of hemoglobin-based blood substitutes focused on improving oxygen uptake and vascular retention. And initially, scientists considered the problem solved. However, new problems continued to arise that reduced the practicality and safety of these hemoglobin-based blood substitutes. Those problems are related to the intrinsic toxicity of hemoglobin.
"You must understand the toxicity factors and find a way to keep the solution from constricting the blood vessels," Simoni said. "Also an effective solution must not produce oxidative stress because that accelerates pro-inflammatory responses and it must be free of pathogens. So it's not a simple project."
Simoni said that many of the blood substitutes under current testing were developed before the intrinsic toxicity problems were identified and have met with limited success. One product has been withdrawn from human trials because it led to an increase in mortality among patients tested. The problems revolve around blood vessel constriction and the pro-oxidant and pro-inflammatory properties of hemoglobin.
The currently tested hemoglobin-based blood substitutes tend to shut down the blood flow in capillaries. While this property helps slow blood loss during a severe cut or hemorrhage, it also prevents blood flow through all tissues in the body when such a blood substitute is used. Another reported disadvantage of solutions under current testing has been the aggravation of oxidative stress and amplification of systemic inflammatory reactions.
"Understanding the limitations of the first generation blood substitutes, some developers have found alternative applications for their existing products, such as treatment of hypotension associated with septic shock, treatment of anemia or a temporary treatment until safe blood could be found. It is clear that such products cannot be used for the treatment of hemorrhagic shock, which is the most important segment of the blood banking industry," Simoni said.
After years of searching, Feola, Simoni and Canizaro have developed a process that makes it possible to create a non-toxic and effective blood substitute product that can be used for the treatment of hemorrhagic shock. The U.S. and international patents awarded to Texas Tech cover the process developed by these university scientists to eliminate hemoglobin toxicity and improve its function as a universal blood substitute. Texas Tech researchers identified the factors that led to toxicity and then developed a purification method that ensures the absolute purity of hemoglobin solutions and a chemical modification method that ensures the lack of toxicity. In addition, the research has led to the development of a blood substitute that has, besides physiological properties of hemoglobin, additional pharmacological activities that effectively eliminate blood vessel constriction, improve the release of oxygen into the body and produce anti-oxidant and anti-inflammatory effects. The solution is also free of bacterial and viral contaminants.
Simoni said that the theoretical basis for their development was in contradiction to the popular theory about blood substitutes, which led others to design an ineffective, even toxic product. "What we have learned is that free hemoglobin-based blood substitutes can no longer be considered inert 'vehicles' for transporting oxygen and carbon dioxide, but they should possess pharmacological properties which can diminish intrinsic toxic effects of hemoglobin and will help eliminate the pathological reactions associated with hemorrhagic shock. Our developed cross-linking agents used in the hemoglobin chemical modification procedure possess these pharmacological properties," he said.
"We have shown that this blood substitute is safe and effective when tested in both animals and humans," Simoni continued. "The effect of the Texas Tech blood substitute was investigated Ôin house’ in a collaboration with many Texas Tech University Health Sciences Center faculty members. Now, research on its therapeutic effect at the molecular level is studied together with Feola, John Griswold, M.D., chairperson of the Department Surgery and Donald Wesson, M.D., chairperson of the Department of Internal Medicine. This product also was tested by independent laboratories in the United States, Europe and Africa and did very well," Simoni said.
The Texas Tech blood substitute is a complex fluid composed of purified bovine hemoglobin, cross-linked intramolecularly with o-adenosine triphosphate (o-ATP) and intermolecularly with o-adenosine and combined with reduced glutathione (GSH).
The patented hemoglobin solution contains physiological properties of hemoglobin and pharmacological properties of ATP, adenosine and GSH that improve blood volume and tissue oxygenation, produce vasodilatory effects and reduce the vasoconstriction that follows hemorrhage, and stimulate the bone marrow to rapidly reproduce natural blood elements. In addition, the blood substitute has anti-oxidant and anti-inflammatory properties and doesn’t result in any toxic reactions. All of these elements work together to create a blood substitute that is possibly the perfect resuscitative fluid for the treatment of acute blood loss, Simoni said.
The scientists chose bovine hemoglobin because it is similar in its molecular structure to human hemoglobin and is a better oxygen carrier than human hemoglobin. The bovine hemoglobin affinity for oxygen is regulated by chlorides rather than 2,3-DPG, as in human hemoglobin. It does not have to be chemically modified with 2,3-DPG analogues in order to unload oxygen. "This initially controversial idea, proposed in the early ‘80s by Dr. Feola, now has wide acceptance in the blood substitute industry," Simoni said.
The substitute also is not susceptible to certain diseases, such as AIDS or hepatitis, which can be transported by human blood, and there is a large-scale availability of bovine erythrocytes. While the recent question of whether human blood can transmit Creutzfeldt’s-Jacob’s disease or its bovine variant is still under study, Simoni said the use of bovine hemoglobin provides a dramatic improvement in blood safety.
"Blood-borne diseases could be avoided by collecting red blood cells from selected healthy cattle. There are plenty of methods to screen bovine blood for prion proteins, which are known to cause mad cow disease (Bovine Spongiform Encephalopathy, BSE). Besides, the developed process might remove the prion proteins from the hemoglobin solution. Texas Tech's hemoglobin chemical modification procedure also can be applied to different types of hemoglobin, such as human, pig or recombinant.
Located in a clean room in the Department of Surgery at the health sciences center is the Blood Substitute Pilot Plant, a working laboratory where the techniques and processes to develop the blood substitute were invented. A blood centrifuge, separation machines, filtration and chromatographic equipment, sanitary pumps, stainless steel sterile vessels and a bioreactor allow the scientists to create small batches of the blood substitute. The facility can produce pure, unmodified and chemically modified hemoglobin solutions in batches of 20 units each, a small amount when compared with the worldwide need of 100 million units. To address this volume, Simoni said that researchers are in the process of finding a way to upscale their blood substitute production. Texas Tech’s blood substitute must still be approved for human trials in the United States, and from there, development of the process into a commercial product will require the construction of a facility capable of producing hundreds or thousands of units of the blood substitute. This next step will require additional funding and support.
"I'm excited about it," said H. Walter Haeussler, Texas Tech's director of technology transfer and intellectual property. "It's significant research and an important contribution to the pharmaceutical industry. However, it's also living proof that if you build a better mousetrap, folks will not beat down a path to your door to embrace it."
Haeussler said that unfortunately only a handful of pharmaceutical companies are considering blood substitute research, and each has its own project under way. This makes it difficult to bring the Texas Tech product to market without a sizable investment from an interested company. He anticipates that it would take upwards of $500 million and as long as 10 years to develop the blood substitute and take it through the human trials required for approval by the Food and Drug Administration.
Haeussler, who over his career has helped bring hundreds of university-developed products to the marketplace through start-up companies, said it is a multi-stage process and he is actively working to help fund the first stage. "It's important to have all the right elements in place, and then we can move forward to begin funding human trials and producing this blood substitute in larger quantities," Haeussler said.
A small company, intent on producing small amounts of the blood substitute and conducting human trials, would cost around $15 million to start, according to Haeussler. This would initiate the project and allow the university to begin the process of proving the effectiveness and safety of the product. Haeussler said eventually a larger pharmaceutical company might invest in the project, especially if Texas Tech can prove it has a commercially and medically acceptable product.
"Once trials begin, it is easier to show a track record of success, which then makes it hard for an established company to ignore," Haeussler said. From there, a larger company might invest in the Texas Tech firm or agree to purchase it and develop the product.
In the meantime, Simoni continues to present papers and showcase the success Texas Tech has had in developing its blood substitute. He says eventually the years of research, testing and refining will provide the world with a safe blood substitute.
"It will be rewarding when we can eliminate the shortages of blood in the world by providing an effective and inexpensive blood substitute that could save millions of people every year. I'm very proud that Texas Tech and Lubbock will have made such a great contribution to this important medical field," Simoni said. "I'm very hopeful that we will make a difference very soon. Blood substitutes are no longer science fiction, they are a reality and a necessity."
Story produced by the Office of Communications and Marketing
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