Kim D. Vandegriff

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (⋅NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in ⋅NO scavenging reactions. All hemoglobins studied exhibited a value of 30 μm −1 s−1 for both⋅NO bimolecular association rate constants and the rate constants for ⋅NO-induced oxidation in vitro. Only the ⋅NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pm and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter⋅NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of ⋅NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.

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.

Colloid osmotic properties of modified hemoglobins: Chemically cross-linked versus polyethylene glycol surface-conjugated

Colloid osmotic pressures of hemoglobin solutions containing unmodified, intramolecularly cross-linked, intermolecularly polymerized, or polyethylene glycol (PEG) surface-conjugated hemoglobin have been measured to determine their macromolecular solution properties. Tetrameric and polymeric hemoglobins show nearly ideal solution behavior: whereas, hemoglobins conjugated to PEG have significantly higher colloid osmotic activity and exhibit solution non-ideality. From these studies, the average calculated molecular weights are 65.300 +/- 3500 for unmodified and intramolecularly cross-linked hemoglobin tetramers, 156,000 for ring-opened raffinose polymerized human hemoglobin, 97,000 for pyridoxalated human hemoglobin conjugated to a carboxy-PEG polymer, and 117,000 for bovine hemoglobin conjugated to a methoxy-PEG polymer. The calculated radius of gyration for tetrameric hemoglobins is 2.9 +/- 0.2 nm compared to 4.9 nm for the polymerized hemoglobin, and 7.2 and 14.1 nm for the human and bovine PEG-conjugated hemoglobins, respectively. Exclusion volumes are calculated to be 823 +/- 148 nm3 for tetramers, 4000 nm3 for polymers, and 13,000 nm3 and 94,000 nm3 for human and bovine PEG-conjugated hemoglobins, respectively. These studies show that polyethylene glycol conjugated to surface amino groups greatly increases the effective macromolecular size of hemoglobin in solution.

CO‐MP4, a polyethylene glycol‐conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats

MP4 (Hemospan) is a Hb‐based oxygen therapeutic agent, based on polyethylene‐glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO‐MP4 on myocardial infarct size following ischaemia and reperfusion in rats.

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.

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.

A Theoretical Analysis of Oxygen Transport: A New Strategy for the Design of Hemoglobin-Based Red Cell Substitutes

In vitro experiments and experiments with artificial capillary systems suggest that transfer of oxygen from cell-free carriers to tissue sites may be faster than it is from red blood cells. On first consideration, this appears to be advantageous, but new data obtained by direct measurements in the microcirculation indicate that facilitated release may actually lead to an autoregulatory decrease in capillary perfusion. This leads to the conclusion that the well-known hemoglobin-oxygen equilibrium curve may not be adequate by itself to describe oxygen delivery by cell-free hemoglobin-based red cell substitutes to achieve optimal tissue oxygenation.

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 ε-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256 kDa, (ii) a molecular radius of ∼6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the “pressor effect” of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

Hemoglobin Oxygen Affinity and the Design of Red Cell Substitutes

A model is described for evaluating a number of properties of cell-free oxygen carriers with regard to their potential to produce autoregulatory vasoactivity in arterioles. The model is based on the Krogh cylinder, an idealized segment of vessel. It is unique because it “maps” the regulatory region of the arterioles - the precapillary segment which senses O2 supply and controls local vascular tone. Through a detailed analysis of the shape and position of oxygen equilibrium curves, the amount of O2 delivered to this sensitive region is analyzed as a function of diffusion, hemoglobin concentration, and P50. The results support the emerging general theory that cell-free oxygen carriers may overcome a diffusive limitation to O2 delivery to tissue, resulting in an over-supply of O2 to regulatory arterioles, leading to local vasoactivity. The model predicts that two potential strategies to overcome this limitation are to lower P50 and to lower the oxygen carrying capacity of the blood.

Advances in Blood Substitutes

To reach cancer cells in a tumor, a blood-borne molecule must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then integrate the resulting information in a unified theoretical framework. Oxygen, one of the smallest blood-borne molecular species, is potentially the most important enhancer of cancer radiation treatment. Its arrival to the needed sites potentially is determined by the unique transport characteristics of the tumor microcirculation. Thus, our analysis and synthesis approach allows us to obtain a better understanding of physiological barriers in solid tumors and to develop novel strategies to exploit and/or overcome these barriers for improved cancer treatment.