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Published by The Vampire Network on the 25th of February 2017True Blood? Not Yet: The Viability of Present and Future Blood Substitutes
Blood substitutes were designed to mimic the properties of whole blood in its ability to increase blood volume and oxygen carrying capacity. There are several products which expand blood volume but do not increase the oxygen carrying capacity. These are used in emergency situations where large volumes of blood are lost, but whole blood transfusion is not an option.
This article will provide an outline of the history of development of “artificial blood” (generation 1 and 2 products), i.e. of synthetic blood substitutes and the currently available (generation 3 products) blood substitutes which increase the oxygen carrying capacity of blood. It will also discuss their usefulness to blood drinkers.
First-generation products (Perfluorocarbon emulsions)
The first generation of synthetic blood substitutes were perfluorocarbon emulsions. Perfluorocarbons are chemically inert molecules capable of dissolving large amounts of many gases, including oxygen. They are hydrophobic, and thus have to be emulsified prior to intravenous (i.v.) administration. Once in the circulation, droplets of this emulsion are taken up by phagocytic cells of the immune system, macrophages, broken down and transported into the lungs. In the absence of significant in vivo metabolism, perfluorocarbons are removed from the body by exhalation.
A major drawback of the perfluorocarbons was that they demonstrated a linear oxygen dissociation curve, in contrast to the sigmoid dissociation curve of blood, which means that oxygen dissociated more easily in the environment where relative oxygen concentration was higher. Thus, most of the oxygen carried by perfluorocarbons was released prior to the arrival of the oxygen-laden molecules into the capillary networks where the need for oxygen delivery is the greatest. Thus, marginal efficacy, low oxygen-carrying capacity, a short effective half-life, temperature instability, as well as serious adverse effects including immune reactions (acute complement activation), disruption of pulmonary surfactant and increased incidence of stroke in Phase III trials, led to this product having been withdrawn from the market.
Second-generation products (Stroma-free hemoglobin)
The second generation of synthetic blood substitutes were based on free (unbound) hemoglobin. Free hemoglobin has been investigated as an oxygen carrier since the 1940s, when researchers realized that it does not elicit an immune response. A solution of free hemoglobin has several advantages over that containing RBCs: it can be sterilized (thus eliminating possibility of infection), and it has a shelf life of approximately 2 years at room temperature. However, free hemoglobin has a higher affinity for oxygen than packed RBCs thus making it less effective at oxygenation when infused i.v. (oxygen molecules are reluctant to detach from the carrier).
This high affinity for oxygen led to other serious complications including kidney dysfunction, increased coagulation (forming of blood cloths) and hypertension (dangerously high blood pressure). Some of these adverse effects were attenuated by various modifications to the hemoglobin molecule to prevent glomerular filtration, thereby avoiding kidney dysfunction, and to stabilize the molecule to withstand heat and chemical purification during production, which prevented aggregation and coagulation. Hypertension induced by infusion of these products, however, was out of proportion to the volume infused and has been more difficult to prevent. It results from free hemoglobin also binding to and sequestering nitric oxide, which is a major factor which normally relaxes blood vessels.
Since then many modifications to the human hemoglobin molecule were tried to reduce its affinity for oxygen and nitric oxide in order to alleviate infusion-related hypertension. Some advances were made, but other just as serious cardiovascular issues arose, including intense vasoconstriction resulting in increased vascular resistance and systemic pressure and reduced cardiac output. Genetically engineered, recombinant variants of human hemoglobin with modifications to decrease their oxygen affinity were tried. The production of these compound has been discontinued due to significant adverse effects including severe vasoconstriction, gastrointestinal distress, fever, chills, and backache. Currently, the idea that unbound hemoglobin could be a viable candidate for an oxygen carrier has been largely abandoned.
Third-generation products (Current)
These are the currently used whole blood substitutes, also known as “oxygen bridges”. All are polymerized hemoglobins (chains of human hemoglobin molecules).
PolyHeme (Northfield Laboratories Inc., Evanston, IL) is a first-generation polymerized hemoglobin made from outdated human blood. It is one of the few products currently being evaluated in a phase III clinical trial that is enrolling patients. It has a half-life of 24 hours, and a shelf life longer than 12 months when refrigerated. An early trial in 2002 showed that 75% of patients with red cell hemoglobin levels less than 1gm/dL survived traumatic injury after receiving PolyHeme as compared to 16% of historical controls (treated with whole blood) at the same hemoglobin level. A later, larger trial showed that 46 of 349 patients treated with PolyHeme died, whereas 35 of 363 patients treated with whole blood died in acute situations indicating that PolyHeme was comparable to whole blood.
There are currently two approved blood substitutes: Oxyglobin and Hemopure. Oxyglobin is approved for use in dogs, and Hemopure for use in humans in South Africa only and under special circumstances on case-by-case basis in other countries including the US and most of Europe.
Hemopure is a polymerized form of bovine hemoglobin with an intravascular half-life of 8-23 hours and a shelf life of 36 months at room temperature. Hemopure is approved in South Africa for the treatment of adult surgical patients who are acutely anemic with the intention of eliminating or reducing the need for RBC transfusions. In the United States, phase II trials have been put on hold. In December 2006, the Blood Products Advisory Committee of the FDA voted against recommending that the US Navy proceed with clinical trials of Hemopure due to the possibility of increased risk of strokes and myocardial infarction. The concern came from initial clinical trials in which increased incidence of stroke, myocardial infarction (MI), acute multi-organ failure (especially kidney) and death was associated with Hemopure vs. whole blood transfusion or blood volume expansion. This is likely due to the tendency of the hemoglobin mimetic, like all blood substitutes before them, to cause a significant increase in blood pressure, by binding, in addition to oxygen, the major vasodilator in humans (nitric oxide). On the background of cardiac and cerebral ischemia (hypoxia) present in both anemia and shock, this can quickly lead to MI, stroke, acute organ failure and death.
However, patients with idiopathic autoimmune hemolytic anemia, which is common among Blood Vampires (vs. non vampires), have been successfully treated with Hemopure.
The newest blood substitute on the market is ErythroMer, developed by Washington University, St. Louis for the Department of Defense (erythromer-blood-substitute). It can be stored in powder form for several months and reconstituted when needed making it highly practical for emergency use. It is currently in clinical trials with no human or large animal data available as of yet. Studies in rodents suggest that the structure of this hemoglobin mimetic may be such that it more closely mimics human hemoglobin in terms of oxygen affinity and binding (loading and unloading), thus minimizing nitric oxide binding, vasoconstriction and dangerous increases in blood pressure (1). Oxygen, nitric oxide and carbon monoxide (also relevant) binding is not identical between rodents and humans; thus, success in rodents does not necessarily translate into Phase II or Phase III clinical trials, leaving the verdict on the clinical usefulness of ErythroMer to the future.
Next-generation blood substitutes
Because of the high incidence of vasoactive side-effects (vasoconstriction and associated hypertension) seen with polymerized hemoglobins, the next generation blood substitutes, which are currently undergoing clinical trials, link hemoglobin with enzymatic carriers. Hemospan (Sangart Inc., San Diego, Calif), also known as MP4OX, is a polyethylineglycol (PEG)-conjugated human hemoglobin currently undergoing clinical trials in the US and Europe. In a small phase II clinical trial in Sweden, Hemospan was as effective and as well-tolerated as control and without negative vasoactive effects. Pyridoxylated hemoglobin polyoxyethylene conjugate (PHP) is a conjugated hemoglobin developed by Apex Bioscience that completed a phase III trial (Identifier: NCT00021502) in August 2009 in patients with shock, likewise with no negative side-effects.
Usefulness to Blood Vampires
All of the currently available blood substitutes and those in clinical trials are hemoglobin-based oxygen carriers, i.e. they are not intact red blood cells (RBCs) and do not have the properties of intact RBCs. They are simply hemoglobin molecules, the oxygen-binding molecules inside RBCs. Because of this characteristic, they are alternatively known as “oxygen bridges”. Furthermore, they are indicated for single use, i.e. once the person has been treated with the blood substitute, they cannot be treated again. This is due to a marked increase in the above mentioned adverse effects (life-threatening hypertension, MI and multi-organ failure, especially kidney failure) with repeated use observed in pre-clinical animal studies. Therefore, these products, although life-saving in emergency situations, remain inferior to whole blood transfusions or transfusions of packed RBCs in chronic situations, such as anemia.
Accordingly, the usefulness of these types of blood substitutes to Blood Vampires, as to non vampires, lie in rare emergency situations, when hemoglobin drops to very low levels.
Challenges to production of “True Blood”
Characteristics of an Ideal Blood Substitute
Characteristics of an ideal blood substitute differ, slightly but significantly, for the requirements of vampires vs. non vampires. For both non vampires and Blood Vampires, an ideal blood substitute should mimic the biological properties of whole blood in its ability to increase blood volume and oxygen carrying capacity, be readily available, with the ability for long-term storage, and substantially decrease or eliminate transmission of blood-borne diseases. In non-vampires, the ideal product should also be universally tolerated without eliciting transfusion rejection reactions or other types of immune system responses, or other major transfusion-associated side-effects (kidney failure).
For Blood Vampires, antigenicity of the substitute is not a concern, as least not in the same way it is for non vampires. Orally ingested blood does not provoke transfusion rejection reactions, probably because the low pH of the stomach removes the blood type-determining antigens from red blood cells (RBCs). However, contrary to non vampires for whom increase in blood volume and oxygen carrying capacity are the only requirements of a blood substitute, vampires may require reconstitution of other, non-cellular components of whole blood from which they may derive benefits, including proteins, co-factors (vitamins and minerals), growth factors, neurotransmitters, etc. Since we do not yet know what these may be, and they may not be the same for all, this presents an additional level of challenges when considering a blood substitute targeted towards the long-term maintenance of health and well-being of Blood Vampires.
Thus, for vampires, the ultimate blood substitute would contain RBCs or hemoglobin and also other contents of RBCs encapsulated in artificial membranes, as well as a cocktail of non-cellular components of blood which are required for optimization.
Practically, this is extremely challenging. Artificial membranes encapsulating hemoglobin and other RBC components which would also escape detection by the immune system are currently out of science’s reach, and whole RBCs, like any other intact cell, cannot be made artificially. The only way to “make” intact RBCs is to differentiate them from human stem cells.
Some studies like this have been initiated, and there have been some positive results; however, these studies are a long way away from clinical trials. Human embryonic stem (hES) cells and the recently established human induced pluripotent stem (hiPS) cells represent potentially unlimited sources of donor-free RBCs, as they can proliferate indefinitely in vitro. Extensive research has been done to efficiently generate transfusable RBCs from hES/iPS cells. Nevertheless, a number of challenges, including obtaining sufficient stem cells and expanding them to make sufficient quantities of RBCs, making “normal” RBCs (with oxygen/carbon dioxide loading/release kinetics identical to human RBCs and low affinity for NO), and navigating around the recipient’s immune system (nobody has yet figured out how to make any cell in culture develop the “coat” of antigens which it would normally develop inside an organism) must be overcome before clinical usage of hES/iPS cell-derived RBCs can become a reality (2, 3). Although the latter is less of a concern for Blood Vampires, the issues of quantity and phenotype of RBCs grown from stem cells are most highly relevant.
The popular press customarily misrepresents the results of these and similar studies and presents them as if the discovery of “true blood” were just around the corner.
It is important to understand that there will be no industrial scale production of limitless supplies of artificial blood any time soon. Least of all will any such product benefit the long-term health and well-being of Blood Vampires in the near future.
References:
- General - http://emedicine.medscape.com/article/207801-overview
- Oxyglobin - http://www.hbo2therapeutics.com/new-page-94/
- Hemopure - http://hemopure.hbo2therapeutics.com
- https://ash.confex.com/ash/2016/webprogram/Paper89042.html
- https://www.ncbi.nlm.nih.gov/pubmed/22648827
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154495/
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