Results of long-term deferiprone (L1) therapy: a report by the International Study Group on Oral Iron Chelators.

This report updates the combined experience of four centres involved in the long-term treatment of transfusional iron overload in 84 patients with the oral iron chelator deferiprone (L1) over 167 patient-years. The source of L1 was variable, including two university research laboratories and three pharmaceutical firms. Compliance was rated as excellent in 48%, intermediate in 36%, and poor in 16% of patients. On a mean L1 dose of 73-81 mg/kg/d, urinary iron excretion was stable, at around 0.5 mg/kg/d, with no indication of a diminishing response with time. Serum ferritin showed a very steady decrease with time from an initial mean +/- 1 SD of 4207 +/- 3118 to 1779 +/- 1154 micrograms/l after 48 months (P < 0.001). 17 patients abandoned L1 therapy. Major complications of L1 requiring permanent discontinuation of treatment included agranulocytosis (three), severe nausea (four), arthritis (two) and persistent liver dysfunction (one). The remaining patients abandoned treatment because of low compliance (three) and conditions unrelated to L1 toxicity (four). Lesser complications permitting continued L1 treatment included transient mild neutropenia (four), zinc deficiency (12), transient increase in liver enzymes (37), moderate nausea (three) and arthropathy (17). There was no treatment-related mortality. Although the complications associated with L1 treatment are significant and require close monitoring, they do not preclude effective long-term therapy in the vast majority of patients. Further well-controlled prospective studies of L1 are required in order to enable proper judgement of its suitability for general long-term clinical use.

Pharmacokinetics of the oral iron chelator deferiprone (L1) in patients with iron overload.

Single oral dose pharmacokinetics of the iron chelator deferiprone (L1) were studied in 24 patients with chronic iron overload and correlated with 24 h urinary iron excretion (UIE) and creatinine clearance. Absorption of L1 was rapid with a t1/2 of 22.2 +/- 17.7 (mean +/- SD) min. The elimination half-life (elt1/2) of the drug was 91.1 +/- 33.1 min and of its metabolite, L1-glucuronide (L1G) 147.7 +/- 52.0 min. Creatinine clearance of the patients correlated significantly with the elimination t1/2 of L1G (r = -0.79, P = 0.002). There was also a significant correlation between 24 h UIE in the 14 patients studied and L1 versus time area under the curve (AUC) (P = 0.007). The total amount of L1 recovered in urine in 24 h comprised 77.9 +/- 13.3% of the L1 dose. L1 efficiency (the 24 h UIE divided by the amount of iron the oral dose of L1 is capable of binding) in the 14 patients was 3.8 +/- 1.9%. These data show for the first time that the urinary elimination of L1G is influenced by the renal function of the patient. Although no significant accumulation of L1 and L1G will occur in most of the patients if L1 is given more than once daily, in some patients with impaired renal function, L1G may accumulate.

Changes in transferrin saturation after treatment with the oral iron chelator deferiprone in patients with iron overload.

AIMS: To evaluate the changes in transferrin saturation in patients with iron overload following the oral administration of the iron chelator deferiprone; to assess the correlation between the degree of transferrin desaturation, the deferiprone dose, and urinary iron excretion. METHODS: Serum samples were obtained from 16 patients with iron overload at different time intervals following the oral administration of deferiprone (50 mg/kg). These samples were analysed using 6M urea/polyacrylamide gel electrophoresis (UPAGE). This method is able to resolve serum transferrin into four different forms (free iron, two forms of monoferric, and diferric). The deferiprone concentration in these samples was estimated using high pressure liquid chromatography (HPLC). Zero time samples (t0) from 10 patients were incubated with 150 microM deferiprone or normal saline either at room temperature or at 37 degrees C for 30 minutes and 24 hours, and also at -20 degrees C for six weeks. Samples were then analysed using UPAGE. RESULTS: A maximum decrease in transferrin saturation from (mean (SD)) 93.0 (10.6)% to 54.5 (17.2)% was observed 72.5 (50.0) minutes after deferiprone administration and in most of the patients coincided with peak deferiprone concentration. This was associated with a maximum rise in the percentage of iron free transferrin (apotransferrin) from 2.9 (7.0)% to 27.3 (17.8)%. The total amount of iron estimated to be removed from transferrin constituted 21.3 (20.2)% of the 24 hour urinary iron excretion measured during the study. When deferiprone (150 mumol/l) was incubated in vitro with t0 samples from 10 patients for 30 minutes and 24 hours at room temperature, 37 degrees C, and at -20 degrees C for six weeks, deferiprone was more efficient at removing iron from transferrin at 37 degrees C, with maximum transferrin desaturation accomplished within 30 minutes compared with 24 hours at room temperature. CONCLUSIONS: The results confirm that deferiprone can remove iron from transferrin when administered orally to patients with iron overload and that transferrin bound iron may, therefore, be a significant source of the iron chelated by deferiprone in vivo.

Oral iron-chelating therapy: the L1 experience.

L1 is the most widely studied oral iron-chelating drug and at present the only one shown to be effective at causing negative iron balance in long-term clinical trials for thalassemia major and other transfusion-dependent refractory anaemias. Because of side-effects, both in experimental animals and in humans, its development as a widely available pharmaceutical agent has been delayed. However, for the large numbers of transfusion-dependent, iron-overloaded patients who do not use DFX because of poor compliance, adverse effects or unavailability of the drug, L1 may be a suitable alternative for iron chelation. However, its use should be restricted to Ethical Committee approved clinical trials. Patients who are capable of using DFX effectively should be encouraged to continue doing so until an oral iron chelator has been fully established for clinical use. It is hoped that 3-hydroxypyrid-4-one analogues of L1 as well as compounds related to pyridoxal isonicotinyl hydrazone, HBED or hydroxamic acid can be found both orally effective and safe for long-term administration. Current and future trials of L1 could address some of the following issues, beside extending present studies on the efficacy and adverse effects of L1: 1. The effect of administering a reduced dose of L1 (< 75 mg/kg per day) on the incidence of adverse effects and on long-term efficacy. 2. The efficacy and adverse effects of L1 at a low dose in patients with non-transfusional iron overload such as thalassaemia intermedia, primary haemochromatosis and congenital haemolytic anaemias. 3. The effect of combining oral L1 with intravenous or subcutaneous DFX on the incidence of adverse effects and efficacy. 4. Elucidation of the mechanisms involved in agranulocytosis and joint toxicity and finding methods to predict for individual susceptibility to these adverse effects and ways of preventing them.

Deferiprone-associated myelotoxicity.

Agranulocytosis developed in a 63-year-old patient with myelodysplasia 6 weeks after commencing treatment with the oral iron chelator deferiprone (L1, 1,2-dimethyl-3-hydroxypyrid-4-one, CP20) at a daily dose of 79 mg/kg. This was the 3rd case of agranulocytosis (neutrophils 0 x 10(9)/l) in clinical trials of L1 at the Royal Free Hospital. The neutrophil count recovered 7 days after stopping L1 and commencing G-CSF at a dose of 300 micrograms daily. Three other patients with milder degrees of neutropenia (neutrophils < 1.5 x 10(9)/l) have also been observed in our trials. The case histories of these 4 patients are described here; other reported cases of neutropenia or agranulocytosis are reviewed. Based on worldwide long-term clinical trials the incidence of agranulocytosis is about 1.6% and of neutropenia 2%.

Zinc concentration in patients with iron overload receiving oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one or desferrioxamine.

AIMS: To determine the changes in serum zinc concentration and the extent of urinary zinc excretion in patients with iron overload receiving the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) or desferrioxamine (DFX), and to correlate these results with blood glucose concentration. METHODS: Serum zinc and ferritin concentrations, urinary zinc and iron excretion were regularly assayed in 39 patients and the glucose tolerance test (GTT) was performed in each patient. Patients were segregated according to their GTT into normal, diabetic, and those with an abnormal GTT. The mean of L1- or DFX associated urinary zinc excretion for each group was determined and compared with the other two groups and with normal value. L1 associated urinary zinc excretion was also compared with L1 dose, serum ferritin values, and urinary iron excretion. RESULTS: Both DFX and L1 were associated with a significantly increased urinary zinc excretion (15.1 (7.3) mumol/24 hours, 11.1 (6.0) mumol/24 hours, respectively) compared with normal subjects. In patients receiving DFX this increase only occurred in patients with diabetes mellitus. Both diabetic and non-diabetic patients receiving L1 treatment excreted more zinc than normal. Diabetic patients receiving L1 or DFX excreted more zinc than non-diabetics receiving the same treatment. No correlation was found between urinary zinc excretion and L1 dose or patients' serum ferritin concentrations. In seven patients receiving long term L1 treatment a fall in serum zinc was observed from an initial 13.6 (1.6) mumol/l to a final 9.6 (0.8) mumol/l. In one patient this was associated with symptoms of dry skin and itchy skin patches requiring treatment with oral zinc sulphate. CONCLUSIONS: In contrast to DFX, L1 treatment is associated with increased zinc loss. This, however, is modest and does not lead in most patients to subnormal serum zinc concentrations. In a few patients whose negative zinc balance may give rise to symptoms, zinc supplementation rapidly corrects the deficit.

Assessment of hepatic iron overload in thalassemic patients by magnetic resonance spectroscopy.

The transverse relaxation time of water protons is shortened by the presence of iron. This shortening depends on the amount and the environment of iron in the sample. We have developed a method for measuring short transverse relaxation time noninvasively by magnetic resonance spectroscopy. To evaluate magnetic resonance spectroscopy as a means of assessing hepatic iron content in patients with transfusional iron overload, we compared the results obtained with this method with those obtained by other means of assessing total body iron content. The correlation between the liver biopsy iron concentration and 1/transverse relaxation time was highly significant (r = 0.95, p < 0.004, n = 6) for iron loads up to 3% dry weight. The correlation between serum ferritin and 1/transverse relaxation time was also significant, but the correlation coefficient was much lower (r = 0.67, p < 0.002, n = 20). The correlation between 24-hr urinary iron excretion and 1/transverse relaxation time was not significant, nor was that between AST and 1/transverse relaxation time. We conclude that magnetic resonance spectroscopic determination of the transverse relaxation time of hepatic water is an accurate method of measuring liver iron content, especially when the iron content is below 3%. Because it is a noninvasive method that is associated with negligible side effects, it could provide clinicians with an excellent means of assessing the effectiveness of the various therapeutic strategies used in the management of patients with iron overload.

Differential toxicity of alpha-keto hydroxypyridine iron chelators and desferrioxamine to human haemopoietic precursors in vitro.

Compliance with iron chelation therapy improves life expectancy in transfusion-dependent haematological disorders. However, failure of compliance with parenteral desferrioxamine (DF) therapy and the expense incurred makes this drug unavailable for most patients in the developing world. We have been evaluating the orally active iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in both preclinical and clinical trials. Five patients have developed reversible agranulocytosis during treatment with this agent. We have now studied the effects of L1, other alpha-ketohydroxypyridines and DF on bone marrow myeloid progenitors using the CFU-GM system. The results show that L1 is less toxic than DF to normal bone marrow myeloid progenitors (ID50:130 mumol/l versus 7.9 mumol/l). The L1 ID50 is within the previously reported range of peak plasma values (80-450 mumol/l). When saturating concentrations of iron were added to the cultures, the mean toxicity of all the chelators was significantly decreased over the range of doses tested, e.g. L1 ID50, 567 mumol/l; DF ID50, > 1000 mumol/l. The toxicity of L1 in vitro was similar for marrows from 3 normal donors and for the recovery marrow from a patient with thalassaemia major who had experienced agranulocytosis. Further studies are required to elucidate the mechanisms of L1-induced agranulocytosis.

Agranulocytosis in a patient with thalassaemia major during treatment with the oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one.

Agranulocytosis developed in a 20-year-old Greek patient with beta-thalassaemia major, 11 weeks after commencing chelation with the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) and 6 weeks after receiving the drug at a total daily dose of 105 mg/kg. The patient presented with generalised weakness, low-grade fever and sore throat. The total white cell count was 2.0 x 10(9)/l with 0.1 x 10(9)/l neutrophils. The patient was admitted to hospital and successfully treated with intravenous broad-spectrum antibiotics. Neutrophil count recovered 7 weeks later. A number of immunological tests were performed in an attempt to elucidate the cause of agranulocytosis. These investigations gave inconclusive evidence for the presence of a weak IgM antibody to myeloid cells exposed to L1 in this patient. Further studies are required, however, to evaluate the mechanism in any other patient who develops agranulocytosis in association with L1 therapy.

Serum non-transferrin-bound iron in beta-thalassaemia major patients treated with desferrioxamine and L1.

Non-transferrin-bound iron (NTBI) in plasma is toxic due to its ability to participate in free radical formation with resultant peroxidation and damage to cell membranes and other biomolecules. NTBI concentration was determined in serum in 12 normal volunteers and in 52 patients with beta-thalassaemia major by a modification of the method described by Singh et al (1990). There was no detectable NTBI in normal individuals. In the patients NTBI values ranged from -1.5 to 9.0 mumol/l (mean +/- SD: 3.6 +/- 2.3). The patients' serum ferritin concentrations ranged from 207 to 11,400 micrograms/l (2674 +/- 2538), total serum iron from 20 to 61 mumol/l (39.5 +/- 9.6) and transferrin saturation from 44 to 110% (84.5 +/- 13.8). The NTBI correlated significantly with serum ferritin (r = 0.467, P < 0.001), total serum iron (r = 0.608, P < 0.001) and transferrin saturation (r = 0.481, P < 0.005). When patients were grouped according to their compliance with desferrioxamine (DFX) therapy, the good compliers had significantly lower NTBI concentrations compared to the poor compliers (poor: 5.4 +/- 1.8 mumol/l v good: 2.7 +/- 1.7 mumol/l, P < 0.001). There was also a significant difference between the level of NTBI and whether or not the patients had complications of iron overload (5.2 +/- 1.7 mumol/l v 2.9 +/- 1.6 mumol/l, P < 0.001). During this study 10 patients were entered into a trial of the oral iron chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1). Their NTBI values were observed during the first 6 months of the trial and showed a significant fall (paired t-test: P = 0.007). These results suggest that the level of NTBI may prove helpful in assessing the efficiency of chelation in patients with transfusion dependent anaemia and help to predict organ damage.

Efficacy and possible adverse effects of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in thalassemia major.

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.