The Development of a
Second-Generation, Designer, Recombinant Hemoglobin
Kenneth E. Burhop
Summary.
The first-generation hemoglobin therapeutics, most of which were created more than 10 years ago, were designed to make a solution that looked identical to hemoglobin contained within a red blood cell. Numerous issues have been raised with their experimental use such as pulmonary and systemic vasoactivity, extravasation of hemoglobin, serum enzyme increases, adverse effects on gastrointestinal motility, generation of myocardial lesions, and potential interactions between hemoglobin and endotoxin. In retrospect, these physiologic effects are not unexpected, since it is now known that an inherent property of all natural (wild-type) hemoglobins is their ability to interact with nitric oxide (NO), secondary to extravasation of the hemoglo- bin into parenchymal tissue.
Key words.
HBOC, Recombinant hemoglobin, Nitric oxide, Blood substitute, Hemoglobin
To address these issues, Baxter has modified the basic hemoglobin function- ality using recombinant technology and site-directed mutagenesis of the distal heme pockets of recombinant human hemoglobin produced in
Escherichia coli, in combination with some of the technology learned fromthe first-generation products (e.g., cross-linking polymerization and deriva- tization to enlarge the size of the molecule). A series of hemoglobin variants with reduced NO reaction rates have been constructed. Substitution of certain amino acids into the heme pockets of a and b-subunits reduced the rate constants for reaction with NO by up to 30-fold relative to wild-type hemo- globin. The systemic hemodynamic responses to these hemoglobins were
127 Medication Delivery, Baxter Healthcare Corporation, DF3-2W, One Baxter Parkway, Deerfield, IL 60015, USA
reduced and the magnitudes of the responses were correlated with the rates of NO scavenging. Therefore, it appears that there are now available viable approaches to modify the intrinsic biologic properties of hemoglobin and produce improved, second-generation hemoglobin products.
Historical Development of Hemoglobin Therapeutics
It is believed that physicians performed the first successful blood transfusion into a human recipient in the seventeenth century, but the practice quickly fell into disfavor when many of the subsequent patients died. While sporadic attempts to transfuse humans were made thereafter, significant progress in the field was made only after scientists gained a better understanding of the critical issues limiting the success of this procedure, such as how to prevent blood from coagulating once it is removed from the body and the fact that humans have different blood types that require all blood units to be tested for compatibility with the intended recipient. In the early part of the twentieth century most of these issues had been resolved so that by the 1920s health- care professionals widely practiced transfusion therapy. Interest in research on blood and blood storage was further stimulated by the need for the treat- ment of military and civilian casualties in World War II, culminating in the establishment of American Red Cross blood banks in 1947. Without question, the implementation of the modern blood banking system has been a major achievement of twentieth century medicine and provided a life saving therapy to millions of patients. On the other hand, like any medical procedure, blood transfusion has several limitations and is not without risk.
In the mid-1980s recognition that serious viral diseases, such as AIDS and
hepatitis, could be transmitted by blood led scientists to intensify their work
to improve the safety of the human blood supply. Subsequent industry-wide
measures have made blood transfusion safer than ever. However, while the
risk of direct transmission of viral diseases by most blood products has been
substantially reduced as a result of increased vigilance in donor screening,
blood testing, and blood processing, the specter of new potential blood borne
pathogens has contributed to ongoing concern about the consequences of
transfusion. In addition, there is mounting evidence that blood transfusion
results in immune suppression that may make patients more susceptible to
common infections such as pneumonia. Other limitations to blood collected
for transfusion include the fact that it can only be stored for six weeks before
it must be discarded and the fact that red cells become impaired during
storage such that the ability to deliver oxygen to tissues immediately after
infusion is reduced. There are also a significant number of red cells (up to
25 %) that become permanently damaged during blood storage such that they
survive in the circulation for only a short period of time.
These issues have spurred government, academic and industry research efforts to find a safe and effective alternative to blood transfusion. The primary focus of this research has been the identification and development of solutions that can perform the oxygen transport function of red blood cells.
While such solutions have often been referred to as “blood substitutes”, this nomenclature is erroneous since these solutions are incapable of performing the other functions of blood such as coagulation. Therefore, these solutions are now more appropriately denoted as oxygen carrying solutions or oxygen therapeutics. The development of such solutions has historically been based on two different technologies that can effectively transport oxygen to tissues, perfluorocarbons and hemoglobins. Perfluorocarbons are synthetic chemicals that readily dissolve gases, while hemoglobins are the pigmented proteins in red blood cells that bind oxygen and enable blood to transport oxygen from the lungs to the tissues. The primary Baxter development effort has been directed to the use of hemoglobin based formulations that are often referred to as hemoglobin based oxygen carriers (HBOCs). Since these formulations may potentially be used to treat conditions for which blood is not used, this class of agents is often referred to as hemoglobin therapeutics.
Researchers have infused solutions containing hemoglobin with varying degrees of success since 1868. However, due to a number of technical limita- tions, and the corresponding lack of success, research in this field languished until the 1970s when it was realized that many of the adverse side effects that plagued early efforts were due to inadequate purification. This in turn led to recognition of the fact that even highly purified hemoglobin does not func- tion well outside of the red cell, unless it is appropriately modified. Fortu- nately, in the late 1970s and early 1980s, a number of modifications were identified that resulted in hemoglobins capable of functioning well when infused into the bloodstream. For the most part, all of these first-generation hemoglobin therapeutics, most of which were created more than 10 years ago, were designed to make a solution that looked identical to hemoglobin con- tained within a red blood cell.
Baxter Hemoglobin Therapeutics
Two first-generation hemoglobin-based oxygen carriers were under develop-
ment by Baxter Hemoglobin Therapeutics and Somatogen during the 1990s
(Diaspirin Cross-Linked Hemoglobin; DCLHb, trade name HemAssist) and a
modified recombinant human hemoglobin (rHb1.1, trade name Optro),
respectively. Each of these products had circumvented the safety concerns
from dimerization of the hemoglobin tetramer by crosslinking the alpha
chains (either chemically in the case of DCLHb or through recombinant engi-
neering with rHb1.1). During the course of their respective development pro-
grams, both the Baxter product and the Somatogen product were entered into clinical trials.
Preclinical and initial clinical studies confirmed their potential utility and safety leading to further clinical development. Specifically, preclinical studies demonstrated that DCLHb and rHb1.1 were well tolerated, non- immunogenic, exhibited excellent oxygen transport properties, were retained in the circulation for clinically useful time intervals, and perfused tissues effi- ciently. Phase I and II studies in a variety of clinical indications found that DCLHb and rHb1.1 were safe and enhanced tissue oxygen consumption and extraction, but had significant vasopressor effects, probably due to nitric oxide (NO) binding. Furthermore, initial evidence of a potential benefit of blood transfusion avoidance and reduction was found in two Phase III surgery trials of DCLHb.
The clinical programs of these two first-generation hemoglobin products merged in 1998 with the acquisition of Somatogen by Baxter. However, the development of both first-generation hemoglobin-based oxygen carriers was discontinued by Baxter in September, 1998 after these products failed to meet the desired clinical safety endpoints. Global concerns about the clinical safety of DCLHb followed the premature termination of the Phase III US Trauma Trial due to an unfavorable imbalance of mortality results. Interestingly, these results were in contrast to the coincident European DCLHb trauma trial (HOST) involving immediate treatment at the scene of the trauma in which there was no significant increase in mortality seen among the patients receiv- ing DCLHb. However, there were infrequently occurring but clinically signif- icant fatal, life-threatening and serious adverse experiences observed across all trials for both DCLHb and rHb1.1 that appeared unusual or seemed to occur more often than expected for their clinical setting. These included Adult Respiratory Distress Syndrome (ARDS), Systemic Inflammatory Response Syndrome (SIRS), Multiple Organ Failures (MOF), acute pancreatitis, and myocardial ischemia.
Initially, it was believed that the vasoconstrictive effects, with the resulting increase in blood pressure seen following infusion of DCLHb or rHb1.1, would not pose a significant problem. In fact, there was considerable evidence suggesting that this pressor effect could be used to a clinical advantage, and that the first-generation products could be used as pharmacologic agents.
Now it appears that the interaction of hemoglobin with nitric oxide and the
physiologic and pathophysiologic consequences of this interaction may be
responsible for many of the adverse effects observed with the first-generation
of purified and modified hemoglobin solutions that were investigated in the
clinic in the 1990s. It was hypothesized that in some vulnerable patients, the
NO binding effects of the hemoglobin may have resulted in vasoconstriction
in certain regional vascular beds, including the mesenteric vasculature,
leading to the development of a cascade of inflammatory effects producing the eventual outcomes noted above.
Therefore, Baxter made a strategic decision to stop development of its first- generation hemoglobin products that possess significant (native) nitric oxide reactivity and to focus efforts on the development of a second-generation recombinant hemoglobin product with reduced nitric oxide scavenging.
Other HBOC Companies
Several hemoglobin-based oxygen carriers are currently undergoing product development. Biopure (Cambridge, MA, USA) is developing Hemopure (bovine hemoglobin that is glutaraldehyde cross-linked to produce a polyhe- moglobin); Northfield (Northfield, IL, USA) is developing Polyheme (hemo- globin from donated human blood that is pyridoxylated to decrease the oxygen binding affinity and glutaraldehyde cross-linked to produce a polyhe- moglobin); and Hemosol (Mississauga, Ontario, Canada) is developing Hemo- link (hemoglobin obtained from donated human blood and O-raffinose crosslinked to produce a polyhemoglobin). All of these agents are in advanced clinical trials (Phase III) and Biopure has recently announced that they have applied for product approval with the FDA. There is limited information about the preclinical and clinical profiles of these products; however, based upon publicly available information, presentations and some publications, these products have generally been reported as effective in a variety of surgical set- tings for avoiding and/or reducing the requirement for allogeneic blood trans- fusions. The safety evaluations reported for some of these products, however, have indicated similar findings to those observed with DCLHb and rHb1.1, including hemodynamic effects (hypertension, vasoconstriction, increased systemic and pulmonary vascular resistance, bradycardia, reduced cardiac output), abdominal discomfort and adverse effects on gastrointestinal motil- ity, skin discoloration, enzyme increases (AST, CK, LDH, lipase), pancreatitis, production of myocardial lesions, and potential interactions with endotoxin.
Baxter’s Second-Generation Recombinant Human Hemoglobin Program
The Baxter Hemoglobin Therapeutics second-generation recombinant hemo-
globin development program is exclusively focused on utilizing recombinant
technology in the bacterium Escherichia coli to produce “designer” hemoglo-
bin solutions. “Recombinant” means that the genetic information for making
a therapeutic protein is inserted into the DNA of a cell such as E. coli. The
DNA then instructs the cell to produce the desired protein, in this case, a new
form of hemoglobin. This technology can be used to induce a variety of cell types to synthesize functional hemoglobin. In addition, modifications of the hemoglobin molecular structure can alter the properties of the molecule, allowing researchers to create hemoglobins with improved functionality or enhanced safety when used as hemoglobin therapeutics.
The second-generation product development effort at Baxter has been driven by the working hypothesis that many of the side effects observed after HBOC infusion are a consequence of the interaction of hemoglobin with NO.
Native hemoglobin interacts very strongly with NO, a ubiquitous and potent chemical messenger found throughout the body and it appears that this scav- enging of NO was associated with some of the adverse outcomes observed with the first-generation hemoglobins. Therefore, Baxter Hemoglobin Thera- peutics developed a hemoglobin-based oxygen carrier with diminished NO scavenging characteristics.
Recombinant technology is the only approach known that can significantly alter the inherent interactions of hemoglobin and NO. Through recombinant technology utilizing site-directed mutagenesis, genes were constructed for several hundred hemoglobin variants. The individual hemoglobin tetramers were internally cross-linked (alpha-alpha fusions) by recombinant engineer- ing. These variants also incorporated amino acid substitutions in the distal heme pockets of both the alpha and beta subunits of hemoglobin leading to steric hindrance for NO entry. The result of this extensive research program was the production of hemoglobin variants with markedly reduced NO reac- tivity that still maintained effective oxygen binding and release. Substitution of certain amino acids into the heme pockets of alpha- and beta-subunits reduced the rate constants for reaction with NO by up to 30-fold relative to wild-type hemoglobin. The systemic hemodynamic responses to these hemo- globins in rats were reduced compared to responses to hemoglobins with wild type NO scavenging rates and the magnitudes of the responses were corre- lated with the rates of NO scavenging. Studies in rats verified that the vaso- constrictive response (increase in mean arterial pressure) corresponded directly to the rate of NO scavenging; i.e., as NO scavenging is decreased the pressor response decreased as did most of the other adverse affects seen with the first-generation hemoglobin solutions (Fig. 1).
Baxter Hemoglobin Therapeutics determined that molecular size and
its impact on extravasation were also important predictors of some of the
adverse pharmacologic effects seen with the hemoglobin molecules; i.e., as
the size of the molecule increased (within certain limits), some of these
adverse effects diminished and circulatory half-life was increased. There-
fore, Baxter Hemoglobin Therapeutics increased the molecular size of the
hemoglobin through polymerization and derivatization of the individual
Fig. 1. Relationship between the blood pressure response in rats and NO scavenging rate of hemoglobin. 350 mg/kg topload doses (n ≥ 6 rats/gp)
Fig. 2. Responses to 2 g/kg topload dose. Bar in each figure indicates duration of infusion
