Oxygen: the poison is in the dose.

Cell-free hemoglobin (Hb) has been blamed for a spectrum of problems, including vasoconstriction pancreatitis, myocardial infarction, and pulmonary hypertension in hemolytic anemia, malaria, and sickle cell anemia, and from Hb-based oxygen carriers (HBOCs). Toxicities have been attributed to scavenging of nitric oxide (NO). However, while NO scavenging may explain many in vitro effects, and some effects in animal models and clinical trials with HBOCs, key inconsistencies in the theory require alternative explanations. This review considers the hypothesis that cell-free Hb oversupplies oxygen to tissues, leading to oxygen-related toxicity, possibly through formation of reactive oxygen species and local destruction of NO. Evidence for this hypothesis comes from various sources, establishing that tissue oxygen levels are maintained over very narrow (and low) levels, even at high oxygen consumption. Tissue is normally protected from excessive oxygen by its extremely low solubility in plasma, but introduction of cell-free Hb, even at low concentration, greatly augments oxygen supply, engaging protective mechanisms that include vasoconstriction and ischemia. The requirement to limit oxygen supply by cell-free Hb suggests novel ways to modify it to overcome vasoconstriction, independent of the intrinsic reaction of Hb with NO. This control is essential to the design of a safe and effective cell-free HBOC.

Cell-free oxygen carriers: scientific foundations, clinical development, and new directions.

The most significant hurdle to the development of a safe and effective hemoglobin-based oxygen carrier ("blood substitute") is generally thought to be its propensity to cause vasoconstriction in the microcirculation and hypertension. Two theories for this effect are currently being studied: in one, scavenging NO by hemoglobin reduces vasorelaxation; in the other, cell-free hemoglobin oversupplies O2 (a known vasoconstrictor) to vascular walls by facilitated diffusion. While both mechanisms might lead to reduction of local NO concentration, the important distinction between the two is that if the NO scavenging theory is correct, it greatly diminishes the prospects to develop any solution based on free hemoglobin. However, if the O2-oversupply theory is correct, modifications to the hemoglobin molecule can be envisioned that can prevent oversupply and reduce toxicity. This review summarizes the development of Hemospan, a novel modification of human hemoglobin whose design is based on the O2-oversupply theory. Because of its low P50 and increased molecular size, the release of O2 in resistance vessels (arterioles) by Hemospan is restricted, and vasoconstriction is greatly reduced.

Hemospan: design principles for a new class of oxygen therapeutic.

Hemoglobin-based oxygen carriers have been under development for decades, but safety concerns have prevented commercial approval. Early designs for modified hemoglobins by polymerization or intramolecular cross-linking reactions increased molecular size and decreased oxygen affinity, but all exhibited side effects of vasoconstriction and reduced blood flow. A new strategy has been established by applying principles of oxygen transport to cell-free hemoglobin. Sangart has developed a new oxygen therapeutic, Hemospan, using site-specific, poly(ethylene) glycol conjugation chemistry designed on two principles: (i) increased macromolecular size to prolong intravascular retention time, and (ii) increased oxygen affinity to prevent premature oxygen offloading in arterioles. In contrast to early-generation products, Hemospan infusion maintains normal arteriolar vascular tone and capillary flow. Phase I and Phase II clinical trials have been completed, showing that Hemospan is well-tolerated in humans, with evidence of efficacy to impart hemodynamic stability in surgical patients under anesthesia. Phase III trials in orthopedic surgery have recently completed enrollment in Europe.

Hemospan improves outcome in a model of perioperative hemodilution and blood loss in the rat: comparison with hydroxyethyl starch.

OBJECTIVES: Hemospan (Sangart Inc, San Diego, CA) (MP4) is a hemoglobin-based oxygen carrier consisting of human hemoglobin modified with polyethylene glycol. This study evaluated the effects of MP4 on blood volume, hemodynamics, and metabolic stability in a rat model of hemodilution and hemorrhage. MP4 was compared with hydroxyethyl starch solutions of differing concentrations (ie, HES 260/0.45 and HES 130/0.4). DESIGN: An open-label, randomized comparison of treatments. SETTING: Pharmaceutical industry. PARTICIPANTS: Sprague Dawley rats. INTERVENTIONS: Rats underwent 50% hemodilution with one of the solutions. Control rats were not hemodiluted. Blood volume was determined at baseline and 0, 60, and 120 minutes after exchange. In separate groups, hemodilution and subsequent 60% hemorrhage were examined to determine effectiveness of hemodilution. MEASUREMENTS AND MAIN RESULTS: Endpoints were blood volume after hemodilution and survival, hemodynamics, and acid-base status during hemorrhage. Volume expansion was similar with MP4 (159% of infused volume) and HES 260/0.45 (145%) and less with HES 130/0.4 (104%). The duration of expansion was longest with MP4 (1-2 hours). In the hemorrhage studies, 2-hour survival was 90% with MP4, 50% with controls, and 10% and 0% with HES 260/0.45 and HES 130/0.4, respectively. The severity of hemodynamic and acid-base changes paralleled the survival, with the least disturbance observed in MP4-treated animals. CONCLUSIONS: Hemodilution with MP4 was more effective in maintaining hemodynamic and metabolic stability than starch solutions or no hemodilution before simulated intraoperative hemorrhage. The benefit of MP4 is not ascribed solely to volume expansion. The results suggest that perioperative administration of MP4 may improve outcomes in surgical settings.

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic.

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic.

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.

Targeted O2 delivery by blood substitutes: in vitro arteriolar simulations of first- and second-generation products.

The O(2) transport from mixtures of commercially produced hemoglobin-based O(2) carriers (HBOCs) and red blood cells (RBCs) flowing through arteriolar-sized (25-mum) conduits is simulated. A generalized treatment of extraluminal O(2) transport processes is used to reflect variations in physiological conditions, such as increased O(2) consumption. Of the HBOCs considered, polymerized bovine hemoglobin (PolyBvHb, p50=54 mmHg), tetrameric cross-linked human hemoglobin (alphaalphaHb, p50=33 mmHg), and PEGylated human hemoglobin (MP4, p50=5 mmHg), only MP4 does not increase O(2) extraction ratios when compared to RBC suspensions alone. A reduction in arteriolar O(2) extraction is likely to be beneficial for HBOCs by preventing O(2)-induced vasoactivity and maximizing the supply of O(2) available to the capillaries. Results from in vivo HBOC transfusion experiments cannot be predicted by the model, unless PolyBvHb has a significant decrease in extraluminal O(2) transport resistance as compared to MP4. This result is consistent with the literature that shows arteriolar O(2) consumption to increase with intravascular pO(2).

Red cell substitutes.

Oxygen-carrying plasma expanders (blood substitutes) have been sought for over a century. Development of current products is a result of evolution in the understanding of proteins in general, of hemoglobin in particular, and of how cell-free hemoglobin interacts with the control of local blood flow to ensure adequate tissue oxygenation. Hemoglobin-based products are considered in four "generations" corresponding to major improvements. First-generation products consisted of hemoglobin, freed of red cell membranes (stroma-free hemoglobin [SFH]), which was renal toxic and vasoactive. Second-generation products were polymerized with aldehyde reagents to reduce or eliminate the renal toxicity, but the products were heterogeneous and still vasoactive. Third-generation products employed more specific intramolecular crosslinking to eliminate polymerization and promote homogeneity, but they also remained vasoactive. Fourth-generation products are based on a new understanding of the way in which microvascular blood flow is controlled and the influence of O(2) delivery to vascular walls. After more than a century of research, one of these new solutions should find use as an alternative to red cells for transfusion in certain clinical settings.

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions.

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.

The role of hemoglobin oxygen affinity in oxygen transport at high altitude.

Hemoglobin is involved in the regulation of O(2) transport in two ways: a long-term adjustment in red cell mass is mediated by erythropoietin (EPO), a response to renal oxgyenation. Short-term, rapid-response adjustments are mediated by ventilation, cardiac output, hemoglobin oxygen affinity (P50), barriers to O(2) diffusion, and the control of local microvascular tissue perfusion. The distribution of O(2) between dissolved (PO2) and hemoglobin-bound (saturation) is the familiar oxygen equilibrium curve, whose position is noted as P50. Human hemoglobin is not genetically adapted for function at high altitude. However, more specialized species native to high altitudes (guinea pig and bar-headed goose, for example) seem to have a lower P50 than their sea level counterparts, an adaptation that presumably promotes O(2) uptake from a hypoxic environment. Humans, native to very high altitude either in the Andes or Himalayan mountains, also can increase O(2) affinity, not because of a fundamental difference in hemoglobin structure or function, but because of extreme hyperventilation and alkalosis.

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates.

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.

Effect of maleimide-polyethylene glycol hemoglobin (MP4) on hemodynamics and acid-base status after uncontrolled hemorrhage in anesthetized swine: comparison with crystalloid and blood.

BACKGROUND: Maleimide-polyethylene glycol hemoglobin, 4.3 g/dL (MP4), is a hemoglobin-based oxygen carrier consisting of native human hemoglobin modified with maleimide polyethylene glycol. This study evaluated resuscitation with MP4 after uncontrolled hemorrhage in anesthetized swine, and compared the effects of MP4 alone with those of standard-of-care crystalloid or crystalloid + blood. METHODS: Pigs were anesthetized with isoflurane and hemorrhaged 250 mL by controlled withdrawal, which was followed by a 5-mm tear in the abdominal aorta. Three groups of pigs (n = 7 each) were randomized after aortic tear to receive 500 mL of MP4, unlimited Ringer's acetate (RA), or 2 L of RA + 250 mL of autologous blood. Measurements included arterial and pulmonary artery pressures, central venous and pulmonary capillary wedge pressures, cardiac output, renal blood flow, urine output, arterial and venous blood gases, lactate concentration, and hemoglobin. Animals were monitored for 150 minutes after hemorrhage. RESULTS: Average body weight (22-24 kg) and hemorrhage volume (28-34 mL/kg) were similar in the three groups. The nadir of mean arterial pressure (23-25 mm Hg) and base excess (-3 to -4 mEq/L) after hemorrhage were similar in all groups, indicating equivalent shock in the three groups. Two hours after administration of MP4, arterial pressure and base excess were significantly improved compared with those of animals administered RA or RA + blood, despite a significantly lower volume of infused fluids (MP4 = 36 +/- 2 mL/kg; RA = 224 +/- 28 mL/kg; RA + blood = 110 +/- 10 mL/kg), and a significantly lower total hemoglobin than achieved with RA + blood. Arterial pressure did not rise above baseline values, cardiac output was not diminished, and systemic vascular resistance was unchanged after administration of MP4, indicating the lack of cardiac effects or peripheral vasoconstriction. CONCLUSIONS: These data demonstrate that resuscitation with a small volume of MP4 induces recovery from hemorrhage without vasoconstriction. Overall, the effects of MP4 alone on hemodynamic and acid-base indices appear to be of greater benefit than those observed with a large volume of RA alone or RA + blood.

Confluent Extended Posterior Left Atrial Wall Ablation: Thinking Inside the Box.

Here, we report intermediate follow-up details after using a technique of confluent posterior left atrial wall epicardial ablation designed to eliminate both existing and future atrial fibrillation (AF) substrates. The method is part of the Convergent hybrid procedure for AF ablation. In this study, multiple confluent epicardial ablations with radiofrequency energy were delivered, spanning the vertical and transverse dimensions of the posterior left atrium, along with facilitated pulmonary vein isolation (PVI). Endocardial mapping and ablation were performed to complete PVI and to ablate the cavotricuspid isthmus. All patients were followed clinically and using two-to-four weeks of continuous monitoring at six, 12, and 24 months, respectively. The average length of follow-up was 488 days. Of the 57 largely unselected patients with persistent or longstanding persistent AF (NPAF), mean duration of AF was 5.6 years. Single procedure freedom from AF through 24 months was 64.5%, and that for all arrhythmias, was 58.9%. Sixty-eight percent of patients were off antiarrhythmic drugs. Four patients (7%) required a second endocardial ablation procedure. A sub-analysis of the observed arrhythmia burden present through follow-up showed this to be small (ie, <1%) in the majority of patients involved in this study. In conclusion, the extended posterior left atrial wall ablation technique discussed here, as part of the Convergent hybrid method, achieved notable single-procedure success in a particularly challenging series of patients with NPAF.

Impact of implantable-cardioverter-defibrillator trials on clinical management of patients with heart failure.

Heart failure is a deadly disease. Every year, tens of thousands of patients die from this condition, many of them suddenly. Efforts aimed at reducing mortality centered initially on antagonizing the neurohormonal system, which is maladaptively upregulated in response to myocardial failure. Antagonists of the renin-angiotensin-aldosterone and adrenergic nervous systems have reduced the rates of cardiovascular mortality and sudden cardiac death. Antiarrhythmic drug therapy has not fared as well. Consequently, efforts to reduce the risk of sudden death have focused on the use of implantable cardioverter-defibrillators (ICDs). How best to identify patients who will benefit from this invasive and expensive therapy has yet to be clearly determined. In this review, we discuss the effectiveness of ICDs in primary and secondary prevention of sudden death in heart failure patients, and examine the impact that the use of ICDs has had on clinical decision making.

WITHDRAWN: PEGylated hemoglobin: Role of surface configuration of PEG for the modulation of hemoglobin vasoactivity.

This paper has been withdrawn since all of the coauthors had not approved of its submission.

A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol.

BACKGROUND AND OBJECTIVES: MP4 (Hemospan), a hemoglobin-based oxygen carrier, has been designed to deliver oxygen to hypoxic tissues without causing vasoconstriction. A phase I clinical trial of MP4 was undertaken to evaluate whether MP4 elicits the clinical side effects associated with previous hemoglobin-based solutions. DESIGN AND METHODS: Twelve volunteers were studied. One cohort (n=4) received 50 mg/kg of MP4, a second (n=4) received 100 mg/kg of MP4, and the third (n=4) received lactated Ringer's solution. Single blind infusions were given at 5 mL/min. Vital signs and symptoms, hematologic parameters, serum chemistry, renal and electrocardiographic measurements were monitored for 15 days after dosing. RESULTS: Five mild adverse events occurred in the controls and 2 each in the 50 mg/kg and 100 mg/kg MP4 groups. None was severe or judged related to MP4 administration by the principal investigator. There were no clinically significant alterations in blood pressure or heart rate, and there were no gastrointestinal symptoms, abdominal or flank pain, loss of appetite or clinically significant alterations of liver or pancreatic enzymes. In one subject (100 mg/kg of MP4), amylase and lipase were slightly above the upper limit of normal 4 hours after dosing, but without associated symptoms or signs. Pharmacokinetic analysis of plasma hemoglobin (assuming no hemolysis) yielded an estimated half-life (T1/2) of 43 hours in the 100 mg/kg MP4 subjects. INTERPRETATION AND CONCLUSIONS: MP4 appears to have a favorable safety profile. Subjects in both study groups survived and did no less well than those in the control group.

Sudden cardiac death: mechanisms, therapies and challenges.

Sudden cardiac death (SCD) is the leading cause of cardiac death in the US. In the past few years, intensive efforts have been made to expand public awareness of SCD and to increase our understanding of its pathophysiology, medical treatment options and device therapy. Significant advances have been made in our ability to prevent SCD in both primary and secondary health care. Two critical issues remain, however: the identification of patients who would benefit from such therapies, and how to achieve even greater prevention, especially primary prevention. The goal of this article is to provide a review of the topic of SCD in the setting of abnormal myocardial substrate, to outline the techniques that are useful in identifying patients at risk, and available treatment options.

Restenosis and drug-eluting stents.

The implantation of intracoronary stents for the treatment of coronary atherosclerotic disease is one of the most common percutaneous procedures. While the procedure brings long-term benefit for a large percentage of patients, a significant number of patients experience in-stent restenosis (ISR). ISR may be caused by a number of biological and procedural factors, including lesion characteristics as well as co-existing disease states like diabetes. Many strategies have been developed to try to reduce the incidence of ISR. The primary methods include systemic pharmacologic treatments, as well as attempts at modifying stents to reduce their role in the development of ISR. Drug-eluting stents are one such modality, and are expected to become a widely used tool in the field of interventional cardiology. This review will focus on the pathophysiology of ISR and possible ways to prevent it, including drug-eluting stents.

Kinetics of NO and O2 binding to a maleimide poly(ethylene glycol)-conjugated human haemoglobin.

The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128-12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the 'R-state' conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.