The idea of using purified Hb as possible universal substitute for red blood cells has been around for almost a century due to Hb's unique oxygen binding property and the lack of blood type antigen. In 1916, Hb was used in human subjects in an attempt to treat anemia. However, such early attempt to use Hb-saline solution within the clinical setting failed due to renal toxicities. It was later determined that early Hb contained erythrocyte membrane stroma lipids that were contaminated with endotoxins, causing sever nephrotoxicity in patients. Consequently, Hb solutions had to be prepared “free” of stromal lipids and endotoxin in order to prevent nephrotoxicities. Two other problems shortly emerged: stroma free Hb had too high an oxygen affinity and too short of an intravascular half in order to be therapeutically useful. Hb had too high of an oxygen affinity because 2,3-DPG normally present in erythrocytic Hb was lost during the purification process. Such a high oxygen affinity did not result in optimal oxygen off-loading in the tissues. In addition tetrameric Hb (a 2 ß 2 ) dissociated into aß dimmers that were filtered by the kidneys and excreted in the urine. Consequently, the HBOCs being clinically tested today have been chemically or genetically “engineered” to produce desirable oxygen offloading characteristics and an extended circulation half-time in order to become a therapeutically useful agent.
Some of the key approaches of hemoglobin oxygen carriers as red blood cell substitutes are illustrated above. Once stroma-free Hb are prepared from human or bovine red blood cells they must be chemically stabilized in order to become therapeutically useful. (A,B) Tetrameric stabilization is accomplished by intramolecular crosslinking between the two a or ß subunits using a site specific crosslinker. (C) The effective molecular weight of Hb can be increased by conjugating it to polyethylene glycol. (D) Polymerized Hb of molecular weights greater than the native Hb tetramer of 64 kDa may be produced through polyfunctional crosslinking agents. (E) Hb can also be encapsulated into liposomes in order to recreate the natural properties of red blood cells.
Preventing the Hb tetramer's dissociation is a major concern in order to suppress renal filtration. Because the alpha/beta (a-ß) dimers are relatively stable, the goal of intramolecular modification is to cross-link the two alpha (a-a) or beta (ß-ß) subunits and stabilize the association of the two alpha/beta (a-ß) dimers. The cross-linking not only prevents tetramer dissociation, but also reduces the affinity of Hb for O 2 . The most popular cross-linkers currently used are DBBF and nor-2-formylpyridoxal 5-phosphate (NFPLP).
Polymerization of Hb through intermolecular cross-linking increases the size of molecules through the formation of Hb oligomers. In the process multiple Hb proteins are linked together through the use of dialdehydes, such as glutaraldehyde and glycoaldeyde. The increase of the size of the oligomers is significant because the molecular weight of the molecule exceeds 500 kDa, compared to a 64.5 kDa unpolymerized Hb tetramers. The increase in size prevents the rapid excretion of the molecule, prolonging the Hb plasma half-life. Unpolymerized Hb tetramers have, however, the unfortunate result of generating excessive viscosity, oncotic pressure, and O 2 affinity. Consequently, it is crucial to obtain high polymerization yields in the manufacturing process. Otherwise, the unpolymerized tetramers must be separated as not to create adverse reactions in the patients. In conclusion, intravascular retention times of HBOCs can be increased by intermolecular crosslinking of stabilized Hbs using crosslinker with poly-functional groups.
Conjugation of Hb is the covalent binding of Hb to a biocompatible polymer, such as polysaccharide, in order to increase its overall size. Such a process achieves similar improvements than those made using polymerization. In a specific case of pegylation, multiple polyethylene glycol (PEG) chains are added to the Hb protein as a means to increasing the molecule's size. It radius, for example, increases from 3 nm to 15 nm once pegylated with 6 PEG chains. Human Hb conjugation with PEG appears to protect the molecule from renal excretion. The intravascular circulation time of a HBOC is extended by conjugating Hb with a macromolecule.
The encapsulation of Hb is based on the idea of recreating the natural properties of RBC without the presence of blood group antigens. Encapsulated Hb is often referred to as “hemosome.” The process of involves the encapsulation of Hb within lipid vesicles using a solution of phospholipids. The encapsulation allows engineers to specify membrane properties of the vesicle. The negatively charged lipids, for example, have demonstrated to limit the aggregation between hemosomes. The alteration of the bilayer membrane may allow for the better diffusion of O 2 and CO 2 .
Genetic engineering is an alternative to chemically modifying Hb. With advances in recombinant DNA technologies, specially modified Hb may be produce from microorganisms, like E. coli or yeast. Prestabilized recombinant human Hb, for example, was produce in E. coli using an expression vector that contained tow mutant globin genes; one had a low oxygen affinity and the other tandemly fused a-globins. Modifications have been made to increase the tetramer's stability and decrease its affinity for O2. Future genetic manipulations may also be able to solve other problems such as the oxidation of Hb into metHb, reaction rate with NO, and short circulation half-life.
The table below characterizes the major difference between transfused red blood cells and infused HBOCs.
Comparison of HBOCs to Transfused Red Blood Cells
The general benefits of HBOCs over transfused red blood cells are summarized below:
The challenges associated with the development of HBOC can be categorized into the following:
HBOCs represent by far the greatest number of products under development in the field of blood substitutes. Most products are currently in Phase III clinical trial which is the final step in safety and efficacy data collection prior to submission for FDA approval.
Three forms of HBOCs are currently in advanced testing stages of the regulatory approval process. One bovine hemoglobin-based solution under development uses the natural low affinity of bovine hemoglobin for oxygen as a major rationale for its use. In addition there are two human hemoglobin-based solutions – one a polymer and the other an oligomeric solution containing a polymeric compound and cross-linked hemoglobin tetramer. These different solutions have different chemical properties as well as different biologic activities. Characteristics of these HBOCs solutions are summarized in the table below.
Comparison of HBOCs:
Hemopure from Biopure, Cambridge , Ma.
Hemopure is a glutaldehyde cross-linked polymer of bovine Hb in which two or more tetramers are covalently linked. Hemopure has been reported to significantly reduce the need for RBCU transfusion undergoing cardiac and aortic surgeries. In addition Hemopure was well tolerated as a resuscitation fluid administered intraopertively. In 2001 Hemopure was approved in South Africa for treatment of adult surgical patients who are acutely anemic and in order to eliminate, reduce, or delay the need for allogeneic RBC transfusion. In October of 2002, Biopure filed a biologic license application (BLA) to the US Food and Drug Administration to market Hemopure in the USA for similar indication in surgical patients than in South Africa . In August 2003, the Food and Drug Administration issued Biopure a response letter requesting additional information concerning Hemopure. Biopure is currently addressing the FDA's questions regarding the product's safety and efficacy. In addition Biopure is conducting additional FDA-requested animal studies. Biopure is also trying to develop Hemopure for other potential medical applications such as in trauma and cardioprotective agent in ischemic conditions.
Hemopure Clinical Trials
Hemolink from Hemosol, Inc, Mississauga , Ontario , Canada .
Hemolink is a polymerized human hemoglobin with residual unpolymerized tetramers. It has completed Phase III trials in cardiothoracic surgery in Canada and is currently ongoing Phase II studies in the same population in the United States . The preliminary studies showed that Hemolink was effective in reducing red blood cell unit (RBCU) requirement in coronary artery bypass graft patients. In early 2003, Hemosol voluntarily suspended a phase II cardiac surgery study when it discovered an imbalance in the incidence of adverse cardiac events occurring in greater numbers in the HemoLink treated group. After electing to terminate the study, Hemosol started investigating the cause of the imbalance, which is thought to be associated with a higher rate of diabetes in the Hemolink treated group.
PolyHeme from Northfield Laboratories, Inc., Evanston , Il .PolyHeme is a glutaldehyde cross-linked polymer of human Hb in which two or more tetramers are covalently linked. In order to replace 2,3-DPG, a pyridoxal molecule is incorporated into each tetramer of PolyHeme to facilitate oxygen offloading. Currently, PolyHeme is being tested in phase III pre-hospital trauma study. The study proposes to demonstrate the safety and efficacy of PolyHeme in improving survival for severely injured bleeding trauma patients. It is important to note the study is being conducted under the informed consent waiver provision, meaning that patients eligible for the study will be unable to provide informed consent. Over 700 patients are being enrolled in the study from over 20 trauma centers across the country. In a separate study PolyHeme has bee shown to be effective in reducing mortality of patients with acute anemia. When compared to an anemic control made up of individual who refused allogeneic RBC transfusion on religious ground, the PolyHeme group had a lower mortality.
PolyHeme Ongoing Phase III Clinical Trial:
Although current formulations appear to cause fewer severe effects, compared to previous products, there remain concerns associated with HBOCs:
Vasoactivity – HBOC products are know to cause vasoconstriction effects . As nitric oxide bind to hemoglobin, nitric oxide becomes less available to cause vascular smooth muscle relaxation. Hence vasoconstricition occurs.
Hemostasis – Studies have shown an increased hemostatic effect in HBOCs due to reversal of the inhibition effect of nitric oxide on platelet aggregation.
Gastrointestinal side effects – Studies have observed gastrointestinal side effects such as nausea, vomiting, diarrhea, and bloating. The binding of nitrous oxide to gastrointestinal intestine tissues is the proposed cause.
Interference with laboratory assays – High concentrations of hemoglobin in plasma due to the infusion of HBOC interferes with laboratory assays. Tests for liver enzymes, bilirubin, amylase and others often yield inaccurate results in results of the presence of HBOCs.