EXCHANGE TRANSFUSION WITH VS -101: A NEW PEGYLATED-HB DESIGNED TO RESTORE PERFUSION AND INCREASE O 2 CARRYING CAPACITY.

Blood products are the current standard for resuscitation of hemorrhagic shock. However, logistical constraints of perishable blood limit availability and prehospital use, meaning alternatives that provide blood-like responses remain an area of active investigation and development. VS-101 is a new PEGylated human hemoglobin-based oxygen carrier that avoids the logistical hurdles of traditional blood transfusion.

How Nitric Oxide Hindered the Search for Hemoglobin-Based Oxygen Carriers as Human Blood Substitutes.

The search for a clinically affordable substitute of human blood for transfusion is still an unmet need of modern society. More than 50 years of research on acellular hemoglobin (Hb)-based oxygen carriers (HBOC) have not yet produced a single formulation able to carry oxygen to hemorrhage-challenged tissues without compromising the body's functions. Of the several bottlenecks encountered, the high reactivity of acellular Hb with circulating nitric oxide (NO) is particularly arduous to overcome because of the NO-scavenging effect, which causes life-threatening side effects as vasoconstriction, inflammation, coagulopathies, and redox imbalance.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers.

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

Background: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage.

Study Design And Methods: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage.

Erythrocytic ATP release in the presence of modified cell-free hemoglobin.

The red blood cell (RBC) has been proposed as an O(2) sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O(2) source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP.

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.

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.

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.

Toxicity and hemodynamic effects after single dose administration of MalPEG-hemoglobin (MP4) in rhesus monkeys.

Maleimide-polyethylene glycol-modified (MalPEG) hemoglobin, 4.3 g/dL (MP4; Hemospan), is a hemoglobin-based oxygen carrier consisting of human hemoglobin (Hb) modified with maleimide polyethylene glycol. This study evaluates the potential toxicity and hemodynamic actions of a single dose of MP4 administered by exchange transfusion to rhesus monkeys.

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.

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge.

Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat.

We have reported a new polyethylene glycol (PEG)-modified, hemoglobin-based O2 carrier (MP4) with novel properties, including a large molecular excluded volume and low PO2 necessary to obtain 50% O2 (approximately 6 Torr). To evaluate the ability of MP4 to transport O2, we compared it with PEG-modified albumin (MPA) using the identical chemistry of attachment of PEG chains. The resulting solutions were well matched with respect to all physical properties except that MP4 is an O2 carrier, whereas MPA is not.

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.

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).

Hemodynamic response and oxygen transport in pigs resuscitated with maleimide-polyethylene glycol-modified hemoglobin (MP4).

Cell-free Hb increases systemic and pulmonary pressure and resistance and reduces cardiac output and heart rate in animals and humans, effects that have limited their clinical development as "blood substitutes." The primary aim of this study was to evaluate the hemodynamic response to infusion of several formulations of a new polyethylene glycol (PEG)-modified human Hb [maleimide PEG Hb (MalPEGHb)] in swine, an animal known to be sensitive to Hb-induced vasoconstriction. Anesthetized animals underwent controlled hemorrhage (50% of blood volume), followed by resuscitation (70% of shed volume) with 10% pentastarch (PS), 4% MalPEG-Hb in lactated Ringer (MP4), 4% MalPEG-Hb in pentastarch (HS4), 2% MalPEG-Hb in pentastarch (HS2), or 4% stroma-free Hb in lactated Ringer solution (SFH).

Probing the conformation of hemoglobin presbyterian in the R-state.

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule.

MP4, a new nonvasoactive PEG-Hb conjugate.

Background: Vasoconstriction has been an obstacle to clinical development of Hb-based O2 carriers. It is proposed that this limitation can be overcome by increasing molecular size and oxygen affinity.

Study Design And Methods: Surface-modified Hb (MP4) was designed, whose properties are consistent with the theory that cell-free Hb engages autoregulatory vasoconstrictive responses to Hb diffusion in the plasma space ("facilitated diffusion").