First human studies with a high-molecular-weight iron chelator.

The release of free, reactive iron from cellular iron stores has been implicated as an important contributor to tissue damage in a variety of clinical situations, including ischemia and reperfusion injury, hemorrhagic shock, and burn injury. Deferoxamine mesylate (DFO), the only iron chelator currently approved for clinical use, is used for the treatment of iron overload, including acute iron poisoning and treatment of chronic iron overload in transfusion-dependent anemias such as beta-thalassemia. However, it is not suitable for acute care situations because of its toxicity, primarily hypotension when given at high intravenous doses, and its short plasma half-life. We have produced a high-molecular-weight iron chelator by chemically coupling DFO to hydroxyethyl starch. This novel chelator (HES-DFO) was administered to healthy male subjects by intravenous infusion over a 4-hour period. The drug was well tolerated, and signs of DFO acute toxicity were not observed. Maximum plasma chelator levels of approximately 3 mmol/L were achieved with HES-DFO, which is more than an order of magnitude higher than has been reported with injections of DFO. Drug residence time in plasma was markedly prolonged, with an initial half-life of 22 to 33 hours. Urinary iron excretion was 7.1 +/- 2.2 mg in 48 hours in the highest dose group, as compared with 0.06 +/- 0.15 mg in control subjects who received normal saline infusions. Intravenous infusion of HES-DFO is well tolerated, produces substantial and prolonged plasma chelator levels, and markedly stimulates urinary iron excretion.

Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy.

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron-deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady-state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf-mediated iron uptake with anti-Tf receptor antibody 42/6, stimulated EPO production in serum-deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES-DFO) and in patients with advanced malignancy treated with anti-Tf receptor antibody 42/6, in a time- and dose-dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

Assessment of microvascular integrity in the isolated perfused rat liver by contrast-enhanced MRI. Attenuation of reperfusion injury by conjugated deferoxamine.

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 hydroxyethyl-starch (HES-DFO).

Protective effects of hydroxyethyl starch-deferoxamine in early sepsis.

The protective effects of hydroxyethyl starch-conjugated deferoxamine (HES-DFO), a macromolecular iron chelator, on the initial pathophysiological cascade in septic shock were evaluated following cecal ligation puncture (CLP) in rats. Animals were given an intravenous dose of 3.0 mL of either vehicle (HES) or HES-DFO immediately following completion of the CLP procedure. Animals were sacrificed 30, 60, 120, and 240 min following CLP, and samples of lung, kidney, bowel, and liver were collected for subsequent analysis of glutathione, myeloperoxidase, and evidence for lipid peroxidation based on measurement of thiobarbituric acid reactive substances and conjugated dienes. In addition, the endotoxin levels were determined in the plasma and histomorphological examination was conducted on tissue samples collected at each time point. At almost all time points, a reduction in lipid peroxidation was noted in the HES-DFO-treated rats (p < .05). Glutathione and myoloperoxidase levels were less affected. Lung tissue from animals receiving HEs demonstrated marked microatelectases, septal destruction, and splicing of basal membranes, which were greatly attenuated in animals having received HES-DFO. Similarly, tubulotoxic and mitochondrial damages observed in kidney samples from HES-treated animals were noticeably reduced in the animals having received the chelator. Liver and gut samples demonstrated unspecific inflammatory injury in both groups of animals. In summary, oxygen radical-mediated tissue damage occurs rapidly following CLP-induced sepsis. Based on histological and biochemical endpoints, treatment with the polymeric iron chelator, HES-DFO, significantly attenuates systemic oxidant injury, the degree of protection being most impressive in the lung and kidney.

Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation.

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.

Hydroxyethyl starch deferoxamine, a novel iron chelator, delays diabetes in BB rats.

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)

Proposed toxic oxidant inhibitors fail to reduce brain edema.

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 post-lesion 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.

Polymer conjugation reduces deferoxamine induced retinopathy in an albino rat model.

PURPOSE: The iron chelating agent deferoxamine mesylate USP (Desferal, Ciba, Summit, NJ) is commonly used in the treatment of acute iron intoxication and chronic iron overload (associated with the transfusion-dependent anemias). When used for prolonged periods of time or in high doses deferoxamine is attended by a range of ocular toxicities. The visual symptoms associated with deferoxamine administration often limit effective iron chelation therapy and can result in permanent vision loss. Deferoxamine has recently been conjugated to certain high molecular weight biocompatible polymers without altering its iron-binding properties. Here the effect of conjugation of deferoxamine to hydroxyethyl starch on retinal toxicity is examined. METHODS: An albino rat model of electroretinographically determined, deferoxamine-induced retinal toxicity has been previously described. We use this model to evaluate and compare both native deferoxamine and hydroxyethyl starch conjugated deferoxamine. RESULTS: Our data show that retinal function, as assessed by the electroretinogram b-wave, is significantly depressed 1 day after a single dose of native deferoxamine, while the b-waves of rats receiving a single dose of hydroxyethyl starch-deferoxamine, are not significantly depressed at any time during the study. In addition, the administered dose of hydroxyethyl starch-deferoxamine resulted in plasma deferoxamine concentrations up to five times greater than those achieved with native deferoxamine. CONCLUSION: These results suggest that hydroxyethyl starch conjugated deferoxamine is associated with less retinal toxicity than native deferoxamine and that it may be a safer alternative for iron chelation therapy.

Role of iron and oxygen radicals in hemorrhage and shock.

This contribution focuses on the role of iron as a critical component in the genesis of oxygen radical mediated tissue injury occurring after global ischemia associated with severe hypovolemic shock. Conventional colloid or crystalloid fluid resuscitation does not adequately protect organs susceptible to reperfusion injury. One approach aimed at attenuating such post-trauma reperfusion injury is systemic, high dose, iron chelation used in combination with colloid fluid replacement.

Failure to alter the course of acute myelogenous leukemia in the rat with subcutaneous deferoxamine.

Deferoxamine (DFO) is an iron chelator that is known to inhibit acute non-lymphocytic leukemia cells in vitro. To explore the possibility that this drug has cytotoxic activity in vivo, rats were inoculated with a small lethal dose (10(2] of tumor cells from the transplantable BN acute myelogenous leukemia model. Animals were then treated with one of several regimens of bolus subcutaneous DFO: 10 mg/day x 5; 20 mg/day x 5; 10 mg/day x approximately 5 weeks; or no DFO. There were no consistently significant differences in survival between any of the DFO and untreated groups. Because the short plasma half-life of DFO was thought to be a potential reason for this lack of protection, a high molecular weight polymeric conjugate of DFO that is known to provide sustained intravascular drug levels was also studied. However, hydroxyethyl starch conjugated with DFO in amounts equivalent to 100 mg free drug (intraperitoneally for 5 days) also failed to have major impact on survival. These findings suggest that it may not be possible to achieve levels of this chelating agent in vivo that are cytotoxic for this disease.

Effects of hydroxyethyl starch conjugated deferoxamine on myocardial functional recovery following coronary occlusion and reperfusion in dogs.

The effect of hydroxyethyl starch-conjugated deferoxamine (HES-DFO) on the recovery of regional myocardial function after 15 min of coronary artery occlusion followed by 3 h of reperfusion of the left anterior descending coronary artery (stunned myocardium) was investigated in anesthetized dogs. Regional myocardial blood flow was measured by radioactive microspheres and regional myocardial segment shortening (%SS) by sonomicrometry. HES-DFO (equivalent of 50 mg/kg DFO), iron saturated HES-DFO (HES-FO), deferoxamine (DFO, 50 mg/kg), or saline were administered by intravenous infusion starting 30 min before occlusion and throughout occlusion. Ischemic bed size and collateral blood flow were similar in all four groups. HES-DFO significantly improved %SS in the ischemic-reperfused region during reperfusion; however, HES-FO and DFO had no effect on %SS as compared to the saline-treated group. HES-DFO and HES-FO had no effect on hemodynamics; however, DFO produced a marked reduction in systemic blood pressure. Since HES-FO had no effect on the recovery of %SS, the beneficial effect of HES-DFO is thought to be due to its iron chelating characteristics. Plasma concentrations of HES-DFO not only reached a higher peak level but also had a longer half life (3 h) than that of regular DFO (20 min). Thus, high-molecular-weight HES-DFO is effective in enhancing the recovery of regional wall motion in stunned myocardium. The reason for the lack of efficacy of DFO compared to HES-DFO at this high dose may be related to the formation of a toxic deferoxamine free radical species.

Hemodynamic effects of intraatrial administration of deferoxamine or deferoxamine-pentafraction conjugate to conscious dogs.

Deferoxamine (DFX) is a specific Fe3+ chelator that is used to manage iron overload, and is being evaluated as an agent to reduce ischemic organ damage that involves iron-mediated OH formation. However, high intravascular doses cause significant hemodynamic changes that may limit or counteract beneficial effects. We used conscious, closed-chest dogs to test the hypothesis that conjugating DFX to pentafraction, a high molecular weight fraction of pentastarch, could reduce such hemodynamic changes. We infused 50 mg/kg of body weight of native DFX, or an equivalent dose as DFX-pentafraction, intraatrially over 15 min. Within 10 min of starting the infusion. DFX increased heart rate from pre-drug values of 105 +/- 11 (mean +/- SEM; N = 9) to 158 +/- 13 beats/min, and reduced left ventricular (LV) systolic pressure from 131 +/- 3 to 99 +/- 16 mm Hg, LV end-diastolic pressure from 12 +/- 3 to 3 +/- 3 mm Hg, and mean arterial pressure (MABP) from 101 +/- 5 to 74 +/- 13 mm Hg. In two dogs, MABP decreased to less than or equal to 35 mm Hg. These parameters returned to predrug values by 60 min after infusion. All of these changes were statistically significant (p less than 0.05). In contrast, infusing DFX-pentafraction (N = 9) caused no significant cardiac or hemodynamic changes other than a transient and slight (approximately 7%) increase in systolic arterial pressures. This conjugate, which prolongs the plasma half-life and does not alter the iron-chelating activity of native DFX, eliminates many undesirable hemodynamic actions. It may be a useful therapeutic alternative to native DFX in some settings.

Quantitative microanalysis of equine synovial fluid glycosaminoglycan concentration.

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 micrograms 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.

High-dose iron-chelator therapy during reperfusion with deferoxamine-hydroxyethyl starch conjugate fails to reduce canine infarct size.

Iron catalyzes reactions during ischemia and reperfusion that contribute to myocardial injury. The iron-chelator deferoxamine blocks these reactions, but undesirable side effects limit the clinical potential of deferoxamine to decrease injury. We tested whether intravenous (i.v.) administration of high doses of a well-tolerated deferoxamine-hydroxyethyl starch (DEFHES) iron-chelator during the last 10 min of ischemia and the first 10 min of reperfusion would decrease canine infarct size. Fourteen chloralose-anesthetized mongrel dogs were randomized to therapy in a blinded fashion with deferoxamine conjugate (75 mg/kg deferoxamine) or hydroxyethyl starch (HES) vehicle alone. Nine other untreated dogs served as controls. Infarct size as a percentage of area at risk (MI/RISK) was not reduced by therapy with deferoxamine conjugate. The deferoxamine dose was five times greater than the maximally tolerated dose of free deferoxamine. Arterial deferoxamine concentrations in the deferoxamine-conjugate group were 0.69 +/- 0.09 mM at onset of reperfusion and 1.37 +/- 0.05 mM at 10 min of reperfusion. Area at risk, ischemic collateral blood flow, and heart rate-blood pressure (HR/BP) product were similar in the groups. Chelation of intravascular iron at the time of reperfusion does not reduce myocardial necrosis in an in vivo model of myocardial ischemia-reperfusion injury.

Abnormal iron distribution in infants of diabetic mothers: spectrum and maternal antecedents.

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.

Modulation of deferoxamine toxicity and clearance by covalent attachment to biocompatible polymers.

A class of high molecular weight iron chelators has been prepared by covalently attaching deferoxamine (DFO), by its amino group, to a variety of biocompatible polymers such as dextran and hydroxyethyl-starch. The iron-binding properties of DFO are virtually unchanged after the attachment procedure, but the toxicity and circulatory half-life are profoundly altered. Competitive iron-binding experiments indicate that the conjugates retain a high affinity for ferric iron. In addition, the derivatives inhibit iron-driven lipid peroxidation as effectively as the parent drug. However, the LD50 in mice (based on DFO equivalents) is approximately 4000 mg/kg for dextran-DFO as compared to 250 mg/kg for free DFO. Consistent with the greatly decreased LD50, intravenous administration of the conjugates in dogs at a dose of 100 mg/kg (body weight) does not cause the severe hypotension associated with intravenous administration of DFO. The plasma half-lives of these adducts are increased greater than 10-fold for dextran-DFO and hydroxyethyl-starch-DFO compared to the free drug. Finally, and most importantly, the conjugates are effective in mediating in vivo iron mobilization and excretion. Because recent evidence implicates iron as an important component of tissue injury in many disease states, these high molecular weight iron chelators may have potential for improved therapy, allowing higher sustained plasma concentrations of the active drug.