iron chelation using subcutaneous infusions of diethylene triamine penta-acetic acid (dtpa)

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Scand J Haematol 1986;36:466-472 Key words: iron overload - thalassaemia - iron chelation - DTPA - desferrioxamine Iron chelation using subcutaneous infusions of diethylene triamine penta-acetic acid (DTPA) M. J. Pippardl, M. J. Jackson2, K. Hoffman3, M. Petrou4 & C. B. Model14 'Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, 2Department of Human Metabolism, School of Medicine, University College, London, U.K., 3Astrapin Pharmaceutical Preparations, Pfaffen-Schwabenheim, West Germany and 4Department of Obstetrics, School of Medicine, University College, London, U.K. The iron chelating ability and potential toxicity of subcutaneous infusions of the calcium and zinc salts of diethylene triamine penta-acetic acid (DTPA) have been assessed in metabolic balance studies in 2 iron-loaded thalassaemic patients. Infusions of calcium DTPA were locally well tolerated and the drug was as effective as desferrioxamine in mobilising iron. However, daily infusions in the 1st patient also produced symptomatic zinc depletion which could not be controlled by simultaneous oral zinc supplements. Zinc DTPA proved ineffective as an iron chelator, but zinc balance could be maintained in the 2nd patient by combining intermittent (every 4 d) use of calcium DTPA with oral zinc supplements. Combined studies with desferriox- amine and calcium DTPA showed the drugs to have additive effects, probably as a result of the chelation of iron from different body sites. Accepted for publication March 4, 1986 CLINICAL BEARING These studies indicate the possibility of devising less toxic schedules of treatment with the relatively unselec- tive, but cheap, synthetic iron chelating agent, calcium DTPA. This might eventually enable the drug to be used to supplement the limited supplies of desferrioxamine in many countries where iron overload due to thalassaemia is common. Thalassaemia major is treated by regular blood transfusion and there is increasing evidence that the consequent, and ultimately fatal, iron over- load may be successfully controlled by regular subcutaneous infusion of the iron chelating agent desferrioxamine (1, 2, 3). However, this drug is expensive and, as a result, is not available in most developing countries where thalassaemia is common. Several new iron chelating agents are active in animals when given by mouth (4, 5), but none has yet undergone large-scale toxicity test- ing in animals, and even if one or more proves safe, effective and inexpensive, none would become available for patient treatment for some years. An interim alternative therapy is therefore needed for existing patients who have no access to desferrioxamine. With this in mind we have investigated the use of subcutaneous infusions of

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Scand J Haematol 1986;36:466-472

Key words: iron overload - thalassaemia - iron chelation - DTPA - desferrioxamine

Iron chelation using subcutaneous infusions of diethylene triamine penta-acetic acid (DTPA)

M. J. Pippardl, M. J. Jackson2, K. Hoffman3, M. Petrou4 & C. B. Model14 'Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, 2Department of Human Metabolism, School of Medicine, University College, London, U.K., 3Astrapin Pharmaceutical Preparations, Pfaffen-Schwabenheim, West Germany and 4Department of Obstetrics, School of Medicine, University College, London, U.K.

The iron chelating ability and potential toxicity of subcutaneous infusions of the calcium and zinc salts of diethylene triamine penta-acetic acid (DTPA) have been assessed in metabolic balance studies in 2 iron-loaded thalassaemic patients. Infusions of calcium DTPA were locally well tolerated and the drug was as effective as desferrioxamine in mobilising iron. However, daily infusions in the 1st patient also produced symptomatic zinc depletion which could not be controlled by simultaneous oral zinc supplements. Zinc DTPA proved ineffective as an iron chelator, but zinc balance could be maintained in the 2nd patient by combining intermittent (every 4 d) use of calcium DTPA with oral zinc supplements. Combined studies with desferriox- amine and calcium DTPA showed the drugs to have additive effects, probably as a result of the chelation of iron from different body sites.

Accepted for publication March 4, 1986

CLINICAL BEARING These studies indicate the possibility of devising less toxic schedules of treatment with the relatively unselec- tive, but cheap, synthetic iron chelating agent, calcium DTPA. This might eventually enable the drug to be used to supplement the limited supplies of desferrioxamine in many countries where iron overload due to thalassaemia is common.

Thalassaemia major is treated by regular blood transfusion and there is increasing evidence that the consequent, and ultimately fatal, iron over- load may be successfully controlled by regular subcutaneous infusion of the iron chelating agent

desferrioxamine (1, 2, 3). However, this drug is expensive and, as a result, is not available in most developing countries where thalassaemia is common. Several new iron chelating agents are active in animals when given by mouth (4, 5 ) , but none has yet undergone large-scale toxicity test- ing in animals, and even if one or more proves safe, effective and inexpensive, none would become available for patient treatment for some years. An interim alternative therapy is therefore needed for existing patients who have no access to desferrioxamine. With this in mind we have investigated the use of subcutaneous infusions of

IRON CHELATION WITH SUBCUTANEOUS DTPA 467

the cheap synthetic iron chelating agent di- ethylene triamine penta-acetic acid (DTPA).

DTPA was developed at the same time as desferrioxamine (6), but it was abandoned because it was painful when administered by i.m. injection (7) and caused various adverse effects (8, 9). It has been suggested that the toxic effects are due to depletion of other essential trace elements, particularly zinc (10). We have pre- viously reported characteristic symptoms of zinc depletion (angular stomatitis, sore eyelids and hair loss) in a transfusion-dependent thalassa- emic boy who was given daily subcutaneous infusions of 1 g of the calcium trisodium salt of DTPA over several weeks (11). We have there- fore investigated the possibility of using the zinc salt of DTPA, which is less toxic in animals than calcium DTPA (12), and whether the zinc defi- ciency associated with regular infusions of cal- cium DTPA can be overcome by oral zinc supplements. We have also examined the effect of combined desferrioxamine and calcium DTPA infusions in removing iron from the body.

Patients and methods The patients were 2 Turkish Cypriots with p thalassaemia major; they were resident in the northern part of Cyprus where desferrioxamine is not freely available. Patient 1 was a boy, 11 yr old who weighed 28 kg. He had been transfused regularly (estimated transfusion iron load was 24 g) and was clinically in good health. His serum ferritin was 9200 wg/l. Patient 2 was a 20-yr-old girl weighing 46 kg who had been transfused regularly. Her serum ferritin was 4720 &I, and iron overload had given rise to hypoparathyroid- ism at the age of 17 yr; this was controlled by oral dihydrotachysterol therapy.

Both patients were studied using conventional meta- bolic balance studies. Diets were planned individually and consisted of identical meals each day, prepared from the same batches of food. This constant diet was admin- istered for a 2 d run-in period, and then for a number of 3, 4 or 5 d balance periods during which all faeces and urine was collected. Faeces samples derived from par- ticular periods were identified by oral administration of carmine red markers taken at the beginning and end of each period. Faecal collections and duplicate diets were homogenised and triplicate aliquots were subjected to

-10

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Zn DTPA lgi12h daily Ca DTPA lg112h daily

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 5 10 15

Days

Figure I . Daily use of zinc and calcium DTPA. 12 h S.C. infusions of 1 g zinc DTPA or calcium DTPA were given daily to patient I . A blood transfusion (2 units) was given immediately before the second 5-d treatment period with calcium DTPA, and raised the Hb from 89 to 120 g/l. Increasing signs of drug toxicity developed from d 14 (see text).

wet acid digestion as previously described (13). The digests and the urines were analysed for their iron, zinc, calcium, magnesium, copper and manganese contents, as were plasma samples taken at regular intervals (before breakfast) throughout the studies. Iron assays were by colour development using bathophenanthroline sulpho- nate and other elements were determined by atomic absorption spectrophotometry.

Calcium DTPA (CaNa, DTPA) and zinc DTPA (ZnNa, DTPA) were both supplied as ampoules con- taining 1 g in 10 ml water for injection (Astrapin Pharmaceutical Preparations, West Germany). Drug infusions were administered using a portable MS16 syringe driver (Graseby Dynamics Ltd., 459 Park Avenue, Bushey, Herts, U.K.). The study was approved by the Ethics Committees of University College Hospital, London, and of the Central Oxford District.

Resu Its Comparison of the zinc and calcium salts of DTPA Patient 1 was treated with zinc DTPA (1 g/d by 12 h S.C. infusion) for one 5-d period, followed

PIPPARD ET AL

-60 -50 -40 -30 -20 -10 0

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+500

+loo0

29 29 29 29 Ca DTPA Ca DTPA Ca DTPA Ca DTPA 0 U 0 0 Blood t r a n s f u s i o n V i t a m i n C 500mg PO dai ly n 4 4 u 4 u

n n Figure 2. Intermittent use of calcium DTPA. 24 h S.C. infusions of 2 g cal- cium DTPA were given to patient 2 during the 1st d of each 4-d balance period. The dietary intake of iron and

and from this baseline the faecal and urinary outputs are plotted separately back towards the zero line. If the resulting value is above the zero line it represents a negative balance and if below a positive balance. A blood trans- fusion (3 units) at the end of the first

€ 5 5 zinc is plotted down from the zero line,

a- m - 20

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l balance period raised the Hb from 85 0 4 8 12 16 to 132 g/l, and ascorbic acid supple-

ments (500 mg by mouth taken in the late evening, away from food) were started between the third and fourth

Days

Faeces 0 U r i n e ----Mean balancelpericd balance periods,

by calcium DTPA at the same dose for 2 further periods. Urinary iron excretion was little affected by zinc DTPA (Figure 1) and the patient was in positive iron balance. In contrast, calcium DTPA therapy produced a marked negative iron bal- ance.

During the second period of calcium DTPA therapy the patient developed a sore mouth and throat, nausea and diarrhoea, sores on both thumbs, and hair loss - toxic effects which have been previously noted with DTPA. Figure 1 dem-

onstrates that the calcium DTPA caused severe zinc depletion (normal range for plasma zinc 11.5-17.5 pmol/l) within 5 d without any effect on the plasma levels of copper, calcium and magnesium. The toxic effects resolved in the 10 d after stopping the calcium DTPA.

Continued therapy using calcium DTPA and zinc sulphate After 2 wk of zinc repletion with oral zinc sulphate (220 mg ZnSO, . 7H,O/d) during

IRON CHELATION WITH SUBCUTANEOUS DTPA 469

-60-

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-40-

G) -30- m -20-

.K u

E" - u c m n 5 -10- L

0-

+lo-

-

Ca DTPA (2g124h) 0

n Desferrioxamine No treatment (2g124h) 0

I I

I I I I I I I I I I I I I 0 4 8 12

Days

a Faeces 0 Urine ----- Mean balancelpe riod

Figure 3. Comparison of equal doses of calcium DTPA and desferrioxamine. Urine and faecal iron excretions were measured after a single 24 h S.C. infu- sion of 2 g of each drug was given to patient 2 at the start of each 4-d bal- ance period. A control period with no drug therapy is also shown.

which patient 1 received no iron chelators, he was started on a combination of calcium DTPA (1 g/d) and oral zinc sulphate (220 mg/d) therapies. Within 48 h the patient complained of perioral paraesthesiae and the chelation was stopped: plasma zinc levels, which had rcturned to normal (13.4 pmol/l) were again slightly below normal (10.6 pmol/l) by the time treat- ment was stopped.

Intermittent use of calcium DTPA with daily oral zinc supplements In an attempt to overcome the toxic effects of daily administration of calcium DTPA seen in the previous studies, patient 2 was treated with daily oral zinc sulphate (220 mg) and 2 g calcium DTPA as a 24 h S.C. infusion on the 1st d of each 4-d balance period. This produced a significant loss of iron in the urine during the day of therapy, which then fell to very low levels during the next 3 d (Figure 2). However, the average loss of iron in each 4-d period was still substantial, ranging from 9.0 to 18.5 mg/d. A similar pattern of excretion of zinc was seen using this therapeu- tic regime. A large amount of zinc was lost from the body immediately following the DTPA therapy, leading to a negative zinc balance for 1-2 d. The zinc balance then became more posi- tive towards the end of each 4-d period. During the first 3 periods the average zinc balance was

negative, but this finally became positive during the 4th period (Figure 2), primarily due to a fall in the faecal zinc output. This overall small nega- tive zinc balance did not cause substantial deple- tion of the body zinc stores since the plasma zinc concentration remained reasonably constant throughout (Figure 2).

Urine manganese excretion was also increased by calcium DTPA (Figure 2). The dietary man- ganese intake was about 60 pmol/d, but we were unable to obtain reliable faecal manganese re- sults. Calcium and magnesium balances re- mained around zero throughout and did not show any consistent changes with calcium DTPA therapy. Urinary copper excretion was increased by calcium DTPA therapy, but this did not affect the overall copper balance.

Comparison of the iron &elating ability of calcium DTPA and desferrioxamine Patient 2 was treated with 2 g calcium DTPA during the 1st d of a 4-d balance period and with 2 g desferrioxamine during the 1st d of the next period (Figure 3). Calcium DTPA was more effi- cient than desferrioxamine at removal of iron by urinary excretion, but did not affect faecal iron levels, whereas desferrioxamine caused loss of iron via both the faeces and urine, the net loss being similar for each drug.

In an attempt to investigate possible interac-

470 PIPPARD ET AL

tions between the two drugs, and whether they were chelating iron from the same source within the body, S.C. infusions of desferrioxamine and calcium DTPA were given separately and in com- bination at doses of equivalent iron-binding capacity (Table 1). The combination of calcium DTPA (3 g/24 h) and desferrioxamine (4 g/24 h) produced an additive effect on iron balance, and the total iron mobilised (173 mg) was comparable to that obtained with 8 g/24 h desferrioxamine (149 mg). Faecal iron excretion with 4 g/24 h desferrioxamine was similar whether the drug was given alone (48.5 mg) or with a calcium DTPA infusion (43.6 mg).

Iron excretion at different doses of calcium DTPA Patient 2 was given increasing doses of S.C. cal- cium DTPA over 24 h periods while taking oral zinc supplements (220 mg ZnSO, . 7H,O/d). Urine iron excretion increased linearly with increasing doses of calcium DTPA (Figure 4). Use of the highest dose (4 g/24 h) was accom- panied by transient mild nausea and fever.

Effects of blood transfusion and vitamin C supplementation In both patients, urine-iron excretion in response to calcium DTPA was slightly decreased follow-

TABLE I Iron excretion wirh S.C. calcium DTPA, desferrioxamine and a combination of both drugs

Therapy Urine Stool Iron iron iron balance (mg) ( w ) (ms)

4 g desferrioxamine/24 h 36.7 48.5 -85.2 3 g calcium DTPA/24 h 61.3 - -61.3 4 g desferrioxamine + 3 g

calcium DTPA/24 h' 129.6 43.6 -173.2 8 g desferrioxamine/24 h 72.0 71.4 -149.4

* Calcium DTPA and desferrioxamine were given simultane-

I6O 1

I I I I I 0 1 2 3 4

Ca DTPA lg124h sc infusion)

Figure 4. Dose response curve for calcium DTPA. Increasing doses of calcium DTPA were given to patient 2 by 24 h S.C.

infusion on alternate days. Urine was collected during and for 24 h after each infusion. Urine iron excretion was complete by 48 h and the values shown are therefore for total urine iron mobilised by each dose.

ing a blood transfusion (Figures 1 and 2). The amount of urine iron mobilised by calcium DTPA after vitamin C supplementation in patient 2 was little changed (Figure 2).

Discussion These studies indicate that calcium DTPA is an efficient iron chelating agent which is locally well-tolerated by subcutaneous infusion. How- ever, zinc depletion was induced in patient l by daily calcium DTPA therapy (Figure 1) without any change in the plasma levels of other ele- ments. We have previously suggested that this adverse effect could be prevented by simul- taneously oral zinc supplements (1 l), but patient 1 developed symptoms of zinc deficiency even during oral zinc therapy. Unfortunately, the potentially less toxic zinc salt of DTPA was ineffective as an iron chelator. These problems - appeared to be overcome in patient 2 where ously using 2 infusion pumps and S.C. injection sites.

Doses were chosen on the basis of equivalent iron binding capacities, assuming one atom to be bound by one molecule of intermittent (every 4 d) use of calcium ' DTPA

ance without the development of clinical features of zinc deficiency and with the plasma zinc level

desferrioxamine as the methane sulphonate = 657). Results are for total iron mobilised over the 3-d balance period. Stool iron excretion is total stool iron minus dietary iron intake.

IRON CHELATION WITH SUBCUTANEOUS DTPA 471

remaining around the lower limit of normal throughout (Figure 2).

Calcium DTPA is known to increase the urin- ary excretion of copper (14) and the present study has now shown increased urine manganese excretion. In the case of copper, the body appears to be able to maintain homeostasis, probably by increasing gastrointestinal copper intake (1 l), but it remains unknown whether manganese depletion could occur. No effect of calcium DTPA therapy was seen on the calcium and magnesium balances.

Calcium DTPA appears to be at least as effi- cient as desferrioxamine in removing iron from the body. The increased iron excretion during DTPA therapy occurs exclusively in the urine, in contrast to desferrioxamine therapy where a con- siderable increase in both faecal and urinary out- put occurs. The faecal excretion of iron after desferrioxamine treatment is known to be de- rived from iron mobilised from hepatocytes into the bile, since iron chelated by desferrioxamine from other body sources is not taken up by the liver and is excreted solely in the urine (13). The lack of effect of simultaneous calcium DTPA infusion on desferrioxamine-induced faecal iron excretion (Table 1) is consistent with the known extracellular distribution of DTPA (15) and with a consequent lack of competition with desfer- rioxamine for iron within hepatocytes. The addi- tive effect of the two drugs may thus result, at least in part, from each chelating iron from dif- ferent sites within the body, as has been sug- gested in rodent studies (16). Neither blood transfusion or vitamin C supplementation had any great effect on urine iron mobilised after calcium DTPA, in contrast to previous studies with desferrioxamine (13, 17). There was a linear response of iron excretion with increasing doses of subcutaneous calcium DTPA (Figure 4), but at the highest doses systemic symptoms (fever and malaise) were noted, similar to those reported with high doses of DTPA given by i.v. infusion

It is clear that calcium DTPA is much less selective for iron than desferrioxamine and can have adverse effects, particularly zinc depletion,

(15).

that do not attend the regular use of desferriox- amine. Nevertheless, our studies suggest that it may be possible to design less toxic schedules of treatment with calcium DTPA which still retain a substantial potential to chelate iron. The safety and efficacy of moderate doses as subcutaneous infusions, given intermittently (e.g. 1-2 times a wk) with oral zinc supplements, now needs to be assessed over a longer period. More general use of calcium DTPA for the treatment of iron over- load in thalassaemia in developing countries would clearly be premature. However, the addi- tive iron chelator effects of calcium DTPA and desferrioxamine could eventually provide a basis for using calcium DTPA to supplement those countries’ meagre supplies of desferrioxamine.

Acknowledgements We are grateful to Mrs. Ruth Rolfe and Miss Hesma Goodburn of the John Radcliffe Hospital, Oxford, UK, for their excellent dietary and technical assistance; to Mr. Bernard Wood of the Whittington Hospital, London, for manganese analyses; and to Dr. T. L. Dormandy and Mr. D. Wickens of the Department of Chemical Pathology, Whittington Hospital, London, for their interest and helpful discussions. Financial support was received from the Wellcome Trust, Lond- on, U.K., the United Kingdom Thalassaemia Society, the Turkish Cypriot Thalassaemia Society, the Sir Hal- ley Stewart Trust, and Prince Sultan bin Abdulaziz of Saudi Arabia.

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IS. Cleton F, Turnbull A, Finch CA. Synthetic chelating agents in iron metabolism. J Clin Invest 1963;42:327-37.

16. Hershko C. A study of the chelating agent diethylenetri- aminepentaacetic acid using selective radioiron probes of reticuloendothelial and parenchymal iron stores. J Lab Clin Med 1975;85:913-21.

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Correspondence to: Dr. M. J . Pippard Section of Haematology Northwick Park Hospital and Clinical Research Centre Watford Road, Harrow Middlesex, HA1 3UJ U.K.