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Evaluation of myocardial iron during iron chelation therapy is not feasible by repeated endomyocardial biopsies owing to the heterogeneity of iron distribution and the risk of complications. Recently, we described a noninvasive method based on magnetic resonance imaging. Here, the method was used for repeated estimation of the myocardial iron content during iron chelation with deferrioxamine in 14 adult nonthalassemic patients with transfusional iron overload. We investigated the repeatability of the method and the relationship between the myocardial iron estimates and iron status. The repeatability coefficient (2sD) was 2.8 micromol/g in the controls (day-to-day) and 4.0 micromol/g in the patients (within-day). Myocardial iron estimates were elevated in 10 of all 14 patients at first examination, but normalized in 6 patients after 6 to 18 months of treatment. If liver iron declined below 350 micromol/g all but one of the myocardial iron estimates were normal or nearly normal. At start (R2 = 0.69, P =.0014) and still after 6 months of iron chelation (R2 = 0.76, P =.001), the estimates were significantly and more closely related to the urinary iron excretion than to liver iron or serum ferritin levels. In conclusion, our preliminary data, which may only pertain to patients with acquired anemias, suggest the existence of a critical liver iron concentration, above which elevated myocardial iron is present, but its extent seems related to the size of the chelatable iron pool, as reflected by the urinary iron excretion. This further supports the concept of the labile iron pool as the compartment directly involved in transfusional iron toxicity.

The role of iron overload as cause of liver dysfunction has never been studied in detail in patients without concomitant hepatotropic infections who receive multiple transfusions. We therefore investigated the relationship between the extent of hepatocellular injury as reflected by serum levels of aminotransferases (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) and several iron status indices in 39 anti-hepatitis C virus-negative (HCV(-)) nonthalassemic patients with transfusional iron overload owing to acquired anemias. In 12 patients, we monitored aminotransferase levels and indices of iron status during iron chelation treatment. Before treatment, elevated aminotransferase activity was seen only at liver iron concentrations more than 300 microM/g. During treatment all aminotransferase values were normal if the liver iron concentration returned below 350 microM/g. At the start of treatment, ALT (R(2) = 0.64, P =.006) and AST activity (R(2) = 0.57, P =.01) were closely related to urinary iron excretion, reflecting the size of the chelatable or the labile iron pool. During treatment, a comparable pattern was seen and the urinary iron excretion was also directly related to the liver iron concentration at concentrations above approximately 400 microM/g. All elevated ALT values were associated with a urinary iron excretion more than 15 mg/24 h. In conclusion, our data suggest the existence of a critical liver iron concentration range, above which hepatocellular injury is seen. The extent of the injury seems to be determined mainly by the size of the chelatable or labile iron pool, supporting the concept of the labile iron pool as the compartment directly involved in iron toxicity. Our findings may be helpful in establishing criteria for safety from complications of transfusional iron overload.

We compared 48-hour urinary iron excretion after a twice-daily subcutaneous bolus injection of deferoxamine and after 12 hours of subcutaneous continuous infusion of the drug in 27 patients with iron overload (mean age, 55.7 years). In most patients, the iron overload was due to multiple transfusions administered during chemotherapy or as part of supportive care for a hematologic or oncologic disorder. One patient had sickle cell anemia and 1 had hereditary hemochromatosis and spherocytosis. Similar urinary iron excretion was observed with the 2 methods of administration; mean +/- SD values were 6935.3 +/- 3832.3 microg/48 hours with subcutaneous bolus injection and 6630.4 +/- 3606.9 microg/48 hours with subcutaneous continuous infusion (P =.3). Twenty-six patients (96.3%) chose to continue therapy with bolus injection. The long-term efficacy of bolus injection was evaluated by measuring the serum ferritin concentration at regular intervals for a follow-up time of 20.1 +/- 4.5 months. Ferritin concentration decreased to below 1000 microg/L in 73% of the patients and to below 500 microg/L in 42% and became normal in 26%. Best results were obtained in patients who were no longer receiving blood transfusions when chelation therapy was initiated. Three of 26 patients (11.5%) had mild, transient side effects after bolus injection. Larger prospective, randomized studies must be conducted before deferoxamine bolus injection can be routinely recommended for patients with iron overload. (Blood. 2000;95:2776-2779)

To further examine the potential clinical usefulness of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) for the chronic treatment of transfusional iron overload, we performed a subchronic toxicity study of the HBED monosodium salt in rodents and have evaluated the iron excretion in primates induced by HBED. The HBED-induced iron excretion was determined for the monohydrochloride dihydrate that was first dissolved in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to the primates either orally (PO) at a dose of 324 micromol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 micromol/kg (37.3 mg/kg, n = 5), sc at 324 micromol/kg (n = 5), and sc at 162 micromol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of 324 micromol/kg (n = 3). In addition, the monosodium salt of HBED in saline was administered to the monkeys sc at a single dose of 150 micromol/kg (64.9 mg/kg, n = 5) or at a dose of 75 micromol/kg every other day for three doses, for a total dose of 225 micromol/kg (n = 4). For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 micromol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO administration of DFO or HBED even at a dose of 300 to 324 micromol/kg was ineffective, the sc injection of HBED in buffer or its monosodium salt, 75 to 324 micromol/kg, produced a net iron excretion that was nearly three times that observed after similar doses of sc DFO. In patients with transfusional iron overload, sc injections of HBED may provide a much needed alternative to the use of prolonged parenteral infusions of DFO. Note: After the publication of our previous paper (Blood, 91:1446, 1998) and the completion of the studies described here, it was discovered that the HBED obtained from Strem Chemical Co (Newburyport, MA) that was labeled and sold as a dihydrochloride dihydrate was in fact the monohydrochloride dihydrate. Therefore, the actual administered doses were 81, 162, or 324 micromol/kg; not 75, 150, or 300 micromol/kg as was previously reported. The new data have been recalculated accordingly, and the data from our earlier study, corrected where applicable, are shown in parentheses.

Fifty-one transfusion-dependent iron-loaded adult patients (38 with thalassemia major) were treated with the orally active iron chelator deferiprone (1,2 dimethyl-3-hydroxypyrid-4-one, L1) at a dose of 75 mg/kg/d (range, 50 to 79). Twenty patients discontinued the drug and five died after a mean of 18.7 months (range, 4 to 35). Of the 20, 5 had arthropathy, 5 had gastrointestinal symptoms, 4 had a rising serum ferritin, 3 had agranulocytosis or neutropenia, 1 had tachycardia, 1 had renal failure, and 1 went abroad. Twenty-six patients continued deferiprone for a mean of 39.4 months (range, 12 to 49). Among these patients, there was no overall significant change in serum ferritin (initial mean, 2,937 microg/L; range, 980 to 5,970; final mean, 2,323 microg/L; range, 825 to 5,970) or in urine iron excretion (initial mean, 31.2 mg/24 h; range, 16.3 to 58. 2; final mean, 32.1 mg/24 h; range, 9.4 to 75.8), implying no overall change in iron stores. When the patients who had received deferiprone for longer than 3 years were considered separately, there was also no significant change in serum ferritin or urinary iron excretion. The initial serum ferritin levels in the 26 patients who continued deferiprone treatment were significantly lower than in those who discontinued the drug (P < .01). The liver iron content in 17 patients who had received deferiprone for 24 to 48 months ranged from 5.9 to 41.2 mg/g dry weight, 50% having levels above 15.0 mg, a level associated with a high risk of cardiac disease due to iron overload. In this study the drug caused fewer side effects and was more effective at maintaining iron status among patients previously well chelated and with lower initial serum ferritin levels.

A comparative study of the iron-clearing properties of subcutaneously administered desferrioxamine B (DFO) with those of orally administered desferrithiocin sodium salt (1), desmethyl desferrithiocin (2), desazadesmethyl desferrithiocin sodium salt (3), desazadesmethyl desferrithiocin pivaloyloxymethyl ester (4), and desazadesmethyl-5,5-dimethyl desferrithiocin (5) in an iron-loaded Cebus monkey model and a non-iron overloaded bile duct-cannulated rat model is presented. All six drugs, which performed well in rodent studies, demonstrated increased efficiency in the Cebus monkey model. When administered to rodents at a daily dosage of 384 mumol/kg over a period of 10 days, drug 1 demonstrated severe renal toxicity. whereas drugs 3, 4, and 5 exhibited severe gastrointestinal (GI) toxicity. Under the same experimental protocol, drug 2 did not show significant toxic side effects. In addition, to further evaluate the iron-clearing properties of analogue 2, a dose-response study was performed in the primates that showed that iron excretion increased in a dose-dependent fashion.

Eleven patients with beta thalassemia major were entered into the trial of the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). Their ages ranged from 17 to 26 years (mean +/- SD, 22.3 +/- 2.7). Six were male and five were female. L1 was administered at an initial daily dose of 42.5 to 60 mg/kg as a single dose. After 4 weeks, the dose was increased to 85 to 119 (102 +/- 10.7) mg/kg for 191 to 352 days divided into either two or four doses daily, except for one patient who developed agranulocytosis after 11 weeks and was taken off the trial. Initial serum ferritin values in the remaining 10 patients ranged between 1,000 and 9,580 (5,549 +/- 3,333) micrograms/L and at end of the trial their mean serum ferritin was significantly lower (4,126 +/- 2,278; P less than .05 using the paired t-test). Urinary iron excretion at a daily dose of 85 to 119 mg/kg administered as two divided doses ranged between 0.14 and 0.82 (0.44 +/- 0.26) mg/kg/24 h. In three patients, the four doses per day schedule caused substantially more iron excretion than the same total dose divided into two. During the course of the trial, several possible adverse effects have been encountered. One patient (female, aged 20) developed agranulocytosis 11 weeks after starting treatment and 6 weeks after beginning treatment with a daily dose of 105 mg/kg. This patient's neutrophil count recovered spontaneously 7 weeks after the discontinuation of L1. A decrease in serum zinc levels to subnormal levels was observed in four patients with symptoms of dry skin, with an itchy scaly rash in two that was associated with low serum zinc levels that responded to zinc therapy. Urinary zinc levels ranged from 4.7 to 23.4 (13 +/- 5.5) mumol/24 h and were above 9 mumol/24 h (upper limit of normal) in eight patients. Mild nausea occurred in three patients and transient diarrhea in a fourth. Mild musculoskeletal symptoms occurred in three patients but settled without discontinuation of L1 therapy in two and with temporary discontinuation of L1 in the third. A transient increase in serum aspartate transaminase was also noted in five patients, but serum aspartate transaminase levels subsequently decreased in all of them. No cardiovascular, neurologic, renal, or retinal toxicities were demonstrable. These results confirm that L1 is an effective oral iron chelator. Further clinical trials are needed to determine the incidence and severity of adverse effects.

In patients with thalassemia intermedia in whom hyperabsorption of iron may result in serious organ dysfunction, an orally effective iron-chelating drug would have major therapeutic advantages, especially for the many patients with thalassemia intermedia in the Third World. We report reduction in tissue iron stores and normalization of serum ferritin concentration after 9-month therapy with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in a 29-year-old man with thalassemia intermedia and clinically significant iron overload (SF 2,174 micrograms/L, transferrin saturation 100%; elevated AST and ALT, abnormal cardiac radionuclide angiogram) who was enrolled in the study with L1 75 mg/kg/day after he refused deferoxamine therapy. L1-Induced 24-hour urinary iron excretion during the first 6 months of therapy was (mean +/- SD, range) 53 +/- 30 (11 to 109) mg (0.77 mg/kg), declining during the last 3 months of L1 to 24 +/- 14 (13-40) mg (0.36 mg/kg), as serum ferritin decreased steadily to normal range (present value, 251 micrograms/L). Dramatic improvement in signal intensity of the liver and mild improvement in that of the heart was shown by comparison of T1-weighted spin echo magnetic resonance imaging with images obtained immediately before L1 administration was observed after 9 months of L1 therapy. Hepatic iron concentration decreased from 14.6 mg/g dry weight of liver before L1 therapy to 1.9 mg/g liver after 9 months of therapy. This constitutes the first report of normalization of serum ferritin concentration in parallel with demonstrated reduction in tissue iron stores as a result of treatment with L1. Use of L1 as a therapeutic option in patients with thalassemia intermedia and iron overload appears warranted.

A comparative study of the iron-clearing properties of subcutaneously (SC) administered deferoxamine (DFO) with those of orally administered 1,2-dimethyl-3-hydroxypyrid-4-one (CP20) and 1,2-diethyl-3-hydroxypyrid-4-one (CP94) is presented. The studies were performed in both a non-iron-overloaded, bile duct-cannulated rat model and an iron-loaded Cebus monkey model. All three drugs performed well in the rodent, promoting the excretion of iron in both the urine and the bile, with total iron output efficiencies of 2.8%, 1.2%, and 7.1%, respectively. The efficiency of DFO increased slightly in the Cebus model, while that of the hydroxypyridones was essentially the same in the monkey, with total iron output efficiencies of 5.5%, 2.1%, and 7.4%, respectively. Iron balance studies showed that both DFO and CP94 were able to maintain the animals in a negative iron balance, while CP20 had little impact.

The mechanism of in vivo iron chelation by 3-hydroxypyridin-4-ones (CP compounds) was studied in hypertransfused rats in which the major storage iron pools in hepatocytes and in the reticuloendothelial (RE) system have been labeled by selective radioiron probes. Both dimethyl-3-hydroxypyridin-4-one (CP 20 or L1) and diethyl-3-hydroxypyridine-4-one (CP 94) have an identical and very high (log beta 3 36) binding constant and selective affinity to iron(III), but the lipid solubility of CP 94 is considerably higher than that of CP 20. Both chelators induced an increase in the fecal excretion of hepatocellular iron with no effect on urinary excretion. In contrast, about one third to one half of the iron mobilized from RE cells was excreted in the urine. The chelating efficiency of CP 20 was comparable with that of deferoxamine (DF), whereas CP 94 was up to eight times more effective than DF. Unlike DF, which had no effect by the oral route, the oral and parenteral effectiveness of both CP compounds was identical. These findings indicate that: (1) lipid solubility is an important determinant of in vivo chelating efficiency; (2) urinary iron excretion induced by the CP compounds is derived from RE cells; (3) part of the iron mobilized from RE cells and all of the iron derived from hepatocytes is excreted through the bile; and (4) contrary to previous observations in cell cultures, there is no in vivo evidence for a diminishing chelating efficiency at the lowest doses used.

Removal of iron from biologically important ligands by desferrioxamine (DF), in vitro, is exceedingly slow because of a kinetic barrier. This kinetic barrier can be overcome by a variety of a small molecular weight anions, of which nitrilotriacetate (NTA) is a prototype. We explored the effect of NTA on DF iron mobilization in vivo. Iron mobilization was increased and the effect was synergistic.

Radioiron excretion in the urine and feces after continuous subcutaneous infusion of desferrioxamine (DF) was studied in normal and hypertransfused rats in whom reticuloendothelial and parenchymal iron stores had been labeled by selective 59Fe probes. The existence of two alternative pathways for the chelation of iron in vivo by DF was indicated by the results. The first pathway was intracellular chelation in hepatocytes with chelated iron excreted through the bile only. The second pathway, which was extracellular, could only be activated after saturation of the transferrin iron binding capacity, with the chelated iron excreted by the kidneys. The last mechanism was probably identical with the mode of action of diethylenetriaminepentaacetic acid (DTPA). Further studies will be required in order to establish whether the enhancement in iron chelation obtained by the continuous infusion of DF in patients with complete saturation of circulating transferrin may or may not be related to the extracellular mechanism of iron chelation in hypertransfused rats.

We have exploited the physiologic mechanism for removal of red cells from the circulation to target the iron chelator desferrioxamine to reticuloendothelial iron stores. Compared with free desferrioxamine injected intravenously, the same dose of desferrioxamine entrapped in resealed red blood cell ghosts resulted in a fourfold to fivefold increase in excretion of radioiron in rats with a selective 59Fe radiolabel of reticuloendothelial iron stores. Desferrioxamine in red cell ghosts did not enhance excretion in rats with selective radiolabeling of parenchymal iron stores. In rats with uniformly radiolabeled iron stores, desferrioxamine in red cell ghosts produced an eightfold to ninefold greater loss of iron in the urine free desferrioxamine intravenously or by slow subcutaneous infusion. Desferrioxamine in red cell ghosts resulted in significantly greater fecal excretion of iron than intravenous desferrioxamine, but desferrioxamine in red cell ghosts and subcutaneous desferrioxamine infusion resulted in similar fecal iron excretion. Clinical application of the red cell ghost method for administration of desferrioxamine and other iron chelators may ber useful for improvement of iron chelation efficiency.

In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total-body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease.