Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P<0.01; total plasma heme: 26.3±4.9 and 87.1±18.4 µM, for CON and HEM, respectively, N=≥6, P<0.001). Interestingly, pre-administration of mice with didox slightly, but significantly, decreased plasma Hb and Heme (plasma Hb: 0.43±0.07 and 0.54±0.07 mg/ml, P<0.05; total plasma heme: 62.7.3±9.5 and 71.5±6.7 µM for i.v. and i.p. didox, respectively, N=6, P<0.05). Concomitantly, acute hemolysis significantly augmented leukocyte (WBC) recruitment and extravasation in the cremaster microcirculation (WBC adhesion: 3.07±0.28 and 13.41±1.02 WBC µm-1, P<0.001; WBC extravasation: 0.72±0.16 and 2.93±0.45 WBC (100x50 µm-2) for CON and HEM, respectively, N=≥5, P<0.001). Didox significantly abrogated this effect of hemolysis, decreasing WBC adhesion to 6.12±0.62 and 2.10±0.30 WBC µm-1 and WBC extravasation to 1.32±0.24 and 1.14±0.21 WBC (100x50 µm-2) (for i.v. and i.p. didox, respectively, N≥5, P<0.001). Flow cytometry demonstrated that hemolysis elevated both the generation of reactive oxygen species (ROS) in the granulocytes of mice and their expression of the Mac-1 integrin subunit, CD11b (ROS: 590±58 and 1140±187 MFI, P<0.01; CD11b: 3028±387 and 4534±416 MFI, for CON and HEM, respect. N=3-6, P<0.01). Importantly, the inhibition of vascular inflammation by didox was associated with a significant decrease in both ROS generation and CD11b expression by granulocytes (ROS: 591±53 and 699±120 MFI, P<0.01; CD11b 2399±126 and 2379±207 MFI, for i.v. and i.p. didox, respectively. N=3-6, P<0.01). Conclusion: Didox, when administrated both intravascularly and intraperitoneally in mice, can inhibit the significant elevations in leukocyte integrin expression and cellular recruitment in the microcirculation that are induced by acute hemolysis. This improvement in vascular inflammation is accompanied by the prevention of oxidative stress in the leukocytes. While didox was found to slightly reduce hemolysis in response to osmotic shock, an interesting observation in itself, it is probable that the beneficial effects of this molecule on vascular inflammation are mediated largely by its effect on oxidative stress parameters. As such, this molecule may provide a novel therapeutic approach to reduce the oxidative stress and vascular inflammation caused by hemolytic events. This includes potential clinical use to treat sickle cell disease and other disorders in which hemolysis is a contributing factor to the pathophysiology.

The accelerating development of potential therapeutic agents for sickle cell anemia (SCA) is welcome and encouraging. Yet, our present comments are prompted by one of these drugs, GBT440,1-5 for which trials in United States and Europe are currently enrolling subjects with SCA. We are somewhat puzzled by the rapid advance of this agent that, in our opinion, has a concerning mode of action that enhances cerebrovascular risk. We here explain this via a linear argument, a simplified (i.e., manageable) model, and an illustrative device. We conclude with specific recommendations to mitigate risk. Other HbS-modifying, affinity-shifting drugs that were previously proposed for SCA (BW12C,6, 7 Tucaresol,8 5-HMF9) also appear to exhibit the same concerning effect we address here. Like GBT440, each forms a Schiff base adduct with globin N-termini, thereby stabilizing hemoglobin its oxy configuration. We use GBT440 as our example because: it is the only such agent now in clinical trials; those studies are enrolling children; its proposed use is to induce ∼30% Hb modification; and relevant data are accessible via three publications1-3 and thirteen meeting posters.4 The purported mechanism of action of GBT440, increasing oxygen affinity, is one of the five categorical strategies that can be used to inhibit HbS polymerization, as recently reviewed by Eaton and Bunn.10 Importantly, they noted that it is difficult to know if the net effect of affinity-shifting agents would be helpful (from decreasing polymerization) or harmful (from compromising oxygen delivery). However, we emphasize that the general concept of affinity-shifting agents can be parsed into different modes of action—and that these have strikingly divergent, and perhaps predictable, risk-to-benefit considerations. A Thought Experiment On Oxygen Delivery We will compare the impacts of two affinity-shifting approaches upon oxygen delivery issues. Recognizing that the complexity of oxygen delivery—from a breath of air to ultimate mitochondrial respiration—renders definitive modeling impossible, we necessarily employ simplifying assumptions that facilitate comparison. All are listed in Table 1, but three are most important. One is that metabolic oxygen demand (and, therefore, oxygen extraction from blood) remains fixed at an average, normal resting value (5 ml O2 per 100 ml blood, i.e., 5 vol%). Another is that both normal and sickle subjects start with whole blood P50 = 27 mm Hg. And another is that the burden of oxygen delivery always must be met without the benefit of increasing microvascular blood flow. The justification for these assumptions, as our starting point, lies in our present primary focus on the brain, as will become apparent later. Nonetheless, farther below we also will describe the impact of re-adding to our model several features of the real sickle milieu. Meanwhile, we suggest that clarity emerges from this simplicity. A Starting Point Given to a normal person, GBT440 (at ∼30% modification) shifts the ODC from curve A to curve E (Figure 1A): P50 ∼18 mm Hg, but biphasic and non-sigmoidal, with a shoulder on the left (the 30% modified, extremely high affinity tetramers) and on the right, emergence of normal oxygen binding by the 70% unmodified tetramers.1 In other words, GBT440 induces the P50 shift because the extreme-affinity component essentially "pulls" the ODC to the left. Interestingly, ODC like curve E are seen in thalassemics with high levels of HbH11 that, like GBT440-modified tetramers, has a myoglobin-like P50, a hyperbolic ODC, without cooperativity or Bohr effect or response to 2,3-DPG.11, 12 The Concept Of Functional Oxygen Content (Foc) To illustrate drug impacts we also plot the oxygen dissociation curves for "Functional Oxygen Content" (FOC; Figure 1B), a device previously used by Bunn and Forget.13 The FOC is what remains of hemoglobin's total oxygen content after the contribution from an extreme-affinity component is subtracted out. So, FOC is the amount of oxygen that can actually be off-loaded at a capillary pO2 that is both achievable and tolerable. They are not the same thing. For skeletal muscle and brain cortex, the critical capillary pO2 threshold is believed to be pO2 ∼15 to 20 mm Hg.14-19 So, myoglobin20 and HbH11,12 and GBT440-modified Hb tetramers1—all of which have P50 ∼2 mm Hg—would be unhelpful for oxygen delivery. (We acknowledge that their contribution would not quite be zero. But a small amount of oxygen unloading from a small proportion of hemoglobin would amount to a very small contribution to oxygen delivery. So we here ignore it in our FOC calculation.) For the normal and sickle patient at baseline, their FOC (Figure 1B) are identical to their ODC (Figure 1A) because all hemoglobin tetramers are fully functional (curves A and B). To assist interpretation of FOC, we display in Table 2 the numerical values for parameters important in sickle cell anemia. Compared to normal (line A), for the anemic sickle patient (line B) to meet metabolic oxygen demand already requires a far lower capillary pO2 and an increased proportionate oxy-to-deoxyHb conversion, with higher ending RBC deoxyHb concentration. Oxygen binding curves. These illustrations of oxygen dissociation curve (ODC, panel A) and functional oxygen content (FOC, panel B) are schematic and intended only to highlight P50 (oxygen affinity) and curve height (amount of bound oxygen, in vol%). These were drawn simply using the graphics function of PowerPoint®. Panel 1A ODC: curve A, normal (Hb=14 g/dl); curve B, sickle baseline (Hb=8 g/dl); curve C, newborn as well as a normal given hypothetical drug "↓DPG"; curve D, HbH and myoglobin and GBT440-modified tetramers; curve E, normal given GBT440 to modify 30% of tetramers; Panel 1B FOC: curve A, normal; curve B, sickle baseline; curve F, sickle given "↓DPG"; curve G, sickle given GBT440 to modify 30% of tetramers. Physiologic Penalties The physiologic implications of the two affinity-shifting strategies are strikingly different. [a] ↓DPG, our imaginary drug, given to the sickle patient shifts FOC from curve B to curve F (Figure 1B), a left shift that retains functionality and entails no loss of maximal oxygen carrying capacity. Per Table 2 (lines B vs F) this does require a further—but manageable—lowering of capillary pO2 to instigate oxygen off-loading, but it does not require any increase in oxy-to-deoxyHb conversion. Yet, this is not without physiological impact. Long ago we demonstrated that a P50 left-shifted from 27 to ∼18 mm Hg is enough to impair maximal exercise ability when ambient oxygen is plentiful.21 Superimposed on a significantly anemic patient, it is quite possible that ↓DPG effects would be noticeable at only moderate exertion levels. On the good side, a left-shift of this magnitude is protective when oxygen availability is limiting,21 as for sickle patients with impaired oxygenation. [b] GBT440, at 30% tetramer modification, eliminates 30% of oxygen delivery capability, superimposed upon the sickle baseline anemia. The net effect is shifting the FOC from curve B to curve G (Figure 1B), with substantial physiologic penalty (Table 2, lines B vs G): further lowering of maximal O2 content, that requires an even lower capillary pO2, to induce a far greater proportionate oxy-to-deoxyHb conversion, resulting in no improvement in the ending deoxyHbS concentration. Of note, this degree of GBT440 modification renders the patient functionally equivalent to having Hb = 5.6 g/dl. Risk Concerns The Perplexing Complexity Of Reality Cerebrovascular Jeopardy The brain comprises ∼2% of body weight but demands ∼20% of oxygen consumption.18 An increase in microvascular flow, requiring arteriolar vasoregulation, is normally its major regulatory solution to insufficient oxygen provision.16 Yet, the systemic endothelial dysfunction of sickle cell anemia impedes normal arteriolar vasoregulation.26 The elevated flow seen in the Circle of Willis, even absent vasculopathic lesions, is deceiving since the sickle patient's brain—like other organs—displays the "perfusion paradox" 27 with macrovascular hyperperfusion but microvascular hypoperfusion. Therefore, sickle patients at baseline already reside in a state of high brain vulnerability, with marginal cerebrovascular blood flow and an impaired ability to supply need.28 Indeed, it is believed that this may underlie their alarming ∼50% lifetime incidence of silent stroke. If a GBT440-like drug is added to the mix, absence of the normal compensatory flow increases would make it challenging to offset the drug-induced reduction of FOC. We, therefore, are concerned that, in sickle context, GBT440-like drugs would impose a state of enhanced cerebrovascular risk. Recommendations Our analysis has assumed GBT440 drug dosing to achieve 30% modification, as proposed by the drug's developers.1 It is possible that there may be a "sweet spot" of lower modification that entails a less unfavorable risk-to-benefit ratio, if there indeed is clinical benefit. Obviously, the balance of benefit and risk can only be truly revealed by clinical studies. But we suggest that these should be advanced with heightened caution. Conclusion We urge our colleagues to be circumspect in application of affinity-modulating drugs in sickle cell anemia—and to recognize that different modes of action may have divergent risk-to-benefit considerations. Clinical studies and care should be conducted with awareness of physiological potential for inadvertent harm, in particular in the vulnerable brain. Silent disease is silent—if not specifically sought, it will not be recognized. And regarding available data, it is paramount to remember that: absence of evidence is not the same thing as evidence of absence. Both authors have advised multiple companies regarding potential therapeutics for sickle disease; neither has a conflict of interest at this time. RPH and BEH wrote this manuscript.

The most widely used drug for iron chelation is deferoxamine (DFO) mesylate. While effective in promoting iron excretion, it requires prolonged daily infusions, often resulting in poor compliance. A clinical trial was conducted using starch-conjugated DFO (S-DFO; 40SD02), a high-molecular-weight iron chelator possessing prolonged vascular retention. Single doses of S-DFO were infused intravenously into groups of four transfusion-dependent patients with beta-thalassaemia at doses of 150, 300, 600 and 900 mg/kg. Urinary iron excretion and various pharmacologic parameters were evaluated for 1 week and safety for 3 weeks. No drug-related effects were observed on clinical chemistries, haematological and coagulation parameters, urinalyses, vital signs or electrocardiograms. Drug-related adverse events were limited to four urticarial reactions, none requiring termination of the infusion. The drug stimulated clinically significant urinary iron excretion, with the highest dose (900 mg/kg) inducing excretion of 1.31 mg of iron/kg (range 0.79-1.90 mg/kg) over 1 week, with residual iron-binding capacity present in the plasma for over 6 d. In summary, treatment with S-DFO, administered weekly, has the potential to achieve iron balance in the poorly compliant patient.

Starch-conjugated deferoxamine (S-DFO) is a long-acting, polymeric iron chelator designed to achieve iron balance in patients requiring frequent transfusions. Results from an ascending single dose study in 16 patients with beta-thalassemia showed that S-DFO is capable of achieving up to 7 days of iron balance (Br. J. Haematol . 138, 374–381, 2007). The multiple dose toxicology study described here was conducted to support extended clinical studies in patients with thalassemia and other iron overload disorders. The study was designed to evaluate physiological effects of S-DFO in iron-loaded animals;1.to seek evidence of cardiovascular side-effects;2.to find evidence of drug accumulation in plasma and other tissues; and3.to determine the No Observable Adverse Effect Level (NOAEL) following biweekly intravenous infusion for 4 weeks.

Reperfusion of an ischemic organ can lead to microcirculatory impairment caused, in part, by the generation of reactive free radicals. The iron-catalyzed formation of these deleterious substances can be counteracted by strong metal chelators like deferoxamine. In this study, the protective effect of deferoxamine conjugate was evaluated by assessment of the hepatic microcirculation in the post-ischemic phase. Assessment of the microvasculature was performed by MRI on the isolated perfused rat liver. The restriction of sinusoids subsequent to reperfusion injury was demonstrated by the use of a particulate superparamagnetic contrast agent trapped in the microvasculature. The protective effect of conjugated deferoxamine was evaluated by both MRI and release of alanine aminotransferase. Contrast-enhanced MRI demonstrated a marked impairment of the microcirculation subsequent to the unprotected reperfusion of the ischemic tissue. This injury was attenuated by deferoxamine conjugated to hydroxyethylstarch (HES-DFO).

Dragsten, Paul R.; Hanson, Gregory J.; Hallaway, Philip E.; Hedlund, Bo E. Author Information

Hydroxyl radicals (.OH) may contribute to beta cell death. Because iron catalyzes .OH production, we examined whether administration of a novel, long-acting iron chelator, hydroxyethyl starch-deferoxamine (HES-DFO) could prevent diabetes in spontaneously diabetic biobreeding (BB) rats. In our colony, a peripheral lymphocyte count (PBLC) < 4200 mm3 has an 88% positive predictive value for onset of diabetes mellitus (DM). Rats with PBLC < 4200 mm3 were randomized at 6 weeks of age to receive 50 mg/kg of HES-DFO (a high molecular weight hydroxyethyl starch-conjugated derivative of deferoxamine) or equimolar hydroxyethyl starch (HES) alone given intraperitoneally three times weekly until DM or 120 days of age. Administration of HES significantly decreased the incidence of IDDM to 57% as compared with the incidence of 87% in the lymphopenic unmanipulated BB rats in the colony (p < 0.01). Administration of HES-DFO further significantly decreased the incidence of IDDM to 31% as compared with the lymphopenic unmanipulated rats (p < 0.01). When analyzed by sex, 3 of 17 (18%) HES-DFO-treated males developed DM, versus 10 of 17 (58%) of HES-treated males (p < 0.05, chi square); 8 of 19 (42%) of HES-DFO-treated females developed DM, versus 11 of 20 (55%) HES-treated females (p = NS). There were no differences between the groups in (1) mean time of onset of DM, (2) serum iron levels at study entry and completion, (3) weekly hematocrits, (4) total lymphocyte counts; and (5) weekly weight gains.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.

Toxic oxidants (oxygen free radicals) have been implicated in the formation of brain edema from ischemia-reperfusion injury or tumor growth. We investigated the ability of an iron chelator, a calcium channel blocker, and a xanthine oxidase inhibitor to reduce formation of brain edema following a cold lesion in cats. The agents were given independently of each other in an attempt to inhibit the Haber-Weiss reaction, prevent Ca++ modulated uncoupling of oxidative phosphorylation, and inhibit the generation of toxic oxidants via xanthine oxidase, respectively. Pentastarch-deferoxamine conjugate at a dose of 50 mg/kg was given 15 minutes before and 60 minutes after the cold lesion. Nimodipine was given at a dose of 1 mg/kg 1 hour before and 2 hours after the cold lesion. Allopurinol was given at a dose of 50 mg/kg 24 hours before, at the time of the lesion and, 24 and 48 hours after the lesion. Gravimetric measurements of multiple brain areas were performed at 24 hours postlesion in the pentastarch-deferoxamine and nimodipine groups and at 72 hours post-lesion in the allopurinol group. None of these agents led to significant reduction in brain edema formation as measured with a gravimetric column of kerosene and bromobenzene. Pentastarch-deferoxamine conjugate was utilized to avoid the confounding effects of arterial hypotension which is seen with intravenous deferoxamine. There was even a suggestion of increased edema in the periventricular white matter in animals treated with nimodipine. Taken together, independent inhibition of the Haber-Weiss reaction, of calcium channels, or of xanthine oxidase does not reduce formation of brain edema in the cold lesion model.

Because chronic hypoxemia causes a redistribution of iron from serum and storage pools into an expanding erythrocyte mass, and because infants of diabetic mothers are often hypoxemic in utero and have a high prevalence of polycythemia at birth, we studied iron distribution in 43 term infants of diabetic mothers. Twenty-four infants were at an appropriate size for gestational age; 19 were large for gestational age. At birth, 28 infants (65%) had abnormal serum iron profiles; eight had decreased ferritin concentrations only (stage 1), nine had decreased ferritin and increased total iron-binding capacity values (stage 2), and 11 had these serum findings plus elevated free erythrocyte protoporphyrin concentrations (stage 3). The hypoglycemic infants who were large for gestational age (n = 14) had a higher prevalence of abnormal iron profiles than euglycemic infants who were appropriate in size for gestational age (n = 20; 93% vs 50%; p = 0.009). Progressively abnormal iron profiles were associated with higher glycosylated fetal hemoglobin values, greater degrees of macrosomia, increased hemoglobin and erythropoietin concentrations, and increased erythrocyte/storage iron ratios. Erythropoietin concentrations were inversely linearly correlated with serum iron values (n = 32, r = -0.54; p = 0.003). The combined erythrocyte and storage iron pools were significantly lower in infants with abnormal iron values whose mothers were diabetic, particularly in infants of women with confirmed diabetic vasculopathy. We speculate that these findings are likely due to (1) increased fetal iron utilization during compensatory hemoglobin synthesis in response to chronic hypoxemia and (2) reduced iron transfer during late gestation complicated by diabetes.

SUMMARY An alcian blue precipitation method for quantifying the hyaluronic acid (ha) and sulphated glycosaminoglycan concentration (sgag) in solutions containing both compounds was assessed. The assay was found to be rapid and reliable in solutions containing 0 to 200 mg of ha/dl and 50 to 1,000 μg of sgag/dl, and was not affected by the presence of protein, hemoglobin, or methemoglobin in concentrations normally found in synovial fluid. The ha and sgag concentrations in intercarpal synovial fluid from 13 clinically normal and 11 arthritic horses were evaluated. A relationship was not found between the concentration of ha and sgag and any other synovial fluid variable. The sgag concentration was found to be markedly high in several of the synovial fluid samples from arthritic horses, but did not correlate with the degree of articular cartilage erosion.

A well but cyanotic newborn was found to have a mutant gamma-globin chain, leading to a functionally abnormal fetal hemoglobin. A single amino acid substitution was found in a site consistent with known adult M hemoglobins. This patient showed no clinical evidence of cyanosis at 5 weeks of age as gamma-chain synthesis was replaced by beta-chain synthesis. A sibling born 20 months later was also cyanotic and the same mutant hemoglobin was found.