annotation iron chelation therapy for iron loaded patients

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BritishJournal of Haematology, 1979.43, 1-5. Annotation IRON CHELATION THERAPY FOR IRON LOADED PATIENTS Widespread iron overload affecting many tissues is a well-recognized feature in the clinical progress of patients treated with regular blood transfusion (Jacobs, 1977). This would be of little account if it were not also associated with degenerative changes, especially in the myocardium, liver and endocrine organs, which appear to be due to iron toxicity and which eventually lead to death, often from cardiac or hepatic failure. Iron chelation therapy with desferrioxamine was introduced in 1963 as a means of removing iron from patients suffering from iron overload but it was not until the study of homozygous p thalassaemic patients by Barry et al(1974) that clear evidence was obtained that long-term parenteral administration of the drug resulted in a reduction in liver iron concentration. Two important observations arise from this work. Firstly, although daily intramuscular injections of desferrioxamine for a 6 year period caused a significant fall in liver iron concentration and serum ferritin levels (Letsky ef al, 1974), the total iron load remained in the grossly pathological range. Secondly, despite the failure to induce normal iron status, liver fibrosis was inhibited, while in the untreated control subjects it invariably progressed. In addition, treated patients had a significantly greater pre-pubertal growth rate. However, this type of therapy is unable to prevent iron loading in the face of regular transfusion and there is no evidence at present that it increases life expectancy. Desferrioxamine is rapidly cleared from the plasma with a half life of 5-10 min (Summers et al, 1979), probably due to a combination of processes including passage into intra- and extracellular spaces (Peters et al, 1966), destruction by an enzyme system present in plasma (Meyer-Brunot & Keberle, 1967) and renal excretion. It has become apparent in recent years that a dose of desferrioxamine given by slow infusion, whether intravenously or subcu- taneously, results in far more effective chelation and iron excretion than when the same dose is given as a single injection (Hussain et al, 1977; Propper et al, 1977; Pippard et al, 1978). It has long been known that iron loaded patients develop a secondary scorbutic state and correction of this by ascorbic acid supplements results in up to threefold increase in iron excretion following desferrioxamine, whether this is given as a single dose (Wapnick et al, 1969) or as an infusion (Hussain et a2, 1977). The amount of iron excreted after a desferrioxamine infusion increases with age (and amount of transfusion) and in many cases this may reach levels of over 100 mg daily. Experience with intramuscular injections suggested that significant amounts of excretion could only be attained after a critical iron load had already accumulated (Modell & Beck, 1974) but use of the infusion technique has shown that negative iron balance may be achieved at any level of loading, and in transfusion-dependent thalassaemics overload might be entirely prevented by early introduction of regular chelation therapy (Pippard et al, 1978). Current results suggest that it is technically possible to overcome the problem of transfu- sional iron overload with the methods now in use, though prophylactic treatment still needs to Heath Park, Cardiff CF4 4XW, Wales. Correspondence: Professor Allan Jacobs, Department of Haematology, Welsh National School of Medicine, OOO7-1048/79/0900-0001$02.00 0 1979 Blackwell Scientific Publications 1

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Page 1: Annotation IRON CHELATION THERAPY FOR IRON LOADED PATIENTS

BritishJournal of Haematology, 1979.43, 1-5.

Annotation

IRON CHELATION THERAPY FOR IRON LOADED PATIENTS

Widespread iron overload affecting many tissues is a well-recognized feature in the clinical progress of patients treated with regular blood transfusion (Jacobs, 1977). This would be of little account if it were not also associated with degenerative changes, especially in the myocardium, liver and endocrine organs, which appear to be due to iron toxicity and which eventually lead to death, often from cardiac or hepatic failure. Iron chelation therapy with desferrioxamine was introduced in 1963 as a means of removing iron from patients suffering from iron overload but it was not until the study of homozygous p thalassaemic patients by Barry et al(1974) that clear evidence was obtained that long-term parenteral administration of the drug resulted in a reduction in liver iron concentration. Two important observations arise from this work. Firstly, although daily intramuscular injections of desferrioxamine for a 6 year period caused a significant fall in liver iron concentration and serum ferritin levels (Letsky ef al , 1974), the total iron load remained in the grossly pathological range. Secondly, despite the failure to induce normal iron status, liver fibrosis was inhibited, while in the untreated control subjects it invariably progressed. In addition, treated patients had a significantly greater pre-pubertal growth rate. However, this type of therapy is unable to prevent iron loading in the face of regular transfusion and there is no evidence a t present that it increases life expectancy.

Desferrioxamine is rapidly cleared from the plasma with a half life of 5-10 min (Summers et a l , 1979), probably due to a combination of processes including passage into intra- and extracellular spaces (Peters et al, 1966), destruction by an enzyme system present in plasma (Meyer-Brunot & Keberle, 1967) and renal excretion. It has become apparent in recent years that a dose of desferrioxamine given by slow infusion, whether intravenously or subcu- taneously, results in far more effective chelation and iron excretion than when the same dose is given as a single injection (Hussain et a l , 1977; Propper et a l , 1977; Pippard et a l , 1978). It has long been known that iron loaded patients develop a secondary scorbutic state and correction of this by ascorbic acid supplements results in up to threefold increase in iron excretion following desferrioxamine, whether this is given as a single dose (Wapnick et a l , 1969) or as an infusion (Hussain et a2, 1977). The amount of iron excreted after a desferrioxamine infusion increases with age (and amount of transfusion) and in many cases this may reach levels of over 100 mg daily. Experience with intramuscular injections suggested that significant amounts of excretion could only be attained after a critical iron load had already accumulated (Modell & Beck, 1974) but use of the infusion technique has shown that negative iron balance may be achieved at any level of loading, and in transfusion-dependent thalassaemics overload might be entirely prevented by early introduction of regular chelation therapy (Pippard et al , 1978).

Current results suggest that it is technically possible to overcome the problem of transfu- sional iron overload with the methods now in use, though prophylactic treatment still needs to

Heath Park, Cardiff CF4 4XW, Wales. Correspondence: Professor Allan Jacobs, Department of Haematology, Welsh National School of Medicine,

OOO7-1048/79/0900-0001$02.00 0 1979 Blackwell Scientific Publications

1

Page 2: Annotation IRON CHELATION THERAPY FOR IRON LOADED PATIENTS

2 A mi ota t ion

be subjected to the rigours of long-term clinical trials. Even so, it is questionable whether the available techniques are acceptable for more than a minority of patients in terms of the social and financial cost. Intramuscular injections are painful and infusions are cumbersome. Doubts have already stimulated research into alternative therapeutic methods.

N e w Iron Chelating Agents The search for biologically active iron chelating drugs has been based primarily on the study

of two experimental models: the inhibition of iron uptake and ferritin synthesis in cultures of Chang liver cells and the induction of iron excretion in the hypertransfused, iron loaded rat. The cell culture system allows an assessment to be made of the availability of intracellular chelatable iron to a candidate drug and a t the same time drug toxicity to the cell can be monitored (White e f al , 1976a). The rat system allows balance studies to be carried out following either oral or parenteral drug administration (Graziano et al , 1974).

Many of the drugs tested are derived from microorganisms that producc low molecular weight chelators as part of their iron transport systems. Most of these are either hydroxamates, like desferrioxamine, or derivatives of 2,3-dihydroxybenzoic acid (2,3-DHB), like enteroche- lin (Neilands, 1977). Graziano e f a / (1974) showed that 2,3-DHB itself was an orally effective chelating agent in rats when given by mouth. Its administration a t a dose of 25 ,ug/kg &hourly produced a sevenfold increase in urinary iron exretion; it was specific for iron and there was no apparent toxicity. A further study of 25 benzoic acid derivatives revealed none more effective than 2,3-DHB in iron loaded rats, though rhodotorulic acid was found to be an effective agent (Grady et al , 1976). Studies with Chang cells showed that a number of substances, though effective chelators, also inhibited protein synthesis (White et a / , 1976b). N'NX-bis-(2,3-dihyd- roxybenzoyl) spermidine is an active chelator which not only removes iron from cells but also detaches it from transferrin (Jacobs et aI , 1977). Tropolone, in addition to removing iron from cultured cells and from transferrin (White et al , 1976~) is a highly effective chelator when administered orally to rats (Grady e t a / , 1978). Unfortunately it is highly toxic both to the cell cultures and to the animals. Grady et a1 (1978) felt that of all the compounds studied for possible oral administration only cholylhydroxamic acid (CHA) was worthy of further study. When given orally to rats it was more effective than desferrioxamine given intraperitoneally though somewhat less effective than rhodotorulic acid. It may be assumed that CHA enters the enterohepatic circulation in a similar way to normal bile acids and removes iron from the liver via the biliary route. It is possible that free CHA in the bile could be reabsorbed. More recently Hoy et al (1979) have shown that the oral administration to rats of an isoniazid- pyridoxal hydrazone at a dose of 100 mg/kg results in an eightfold increase in faecal iron excretion.

The new drugs under investigation a t the present time fall into three groups. Firstly, improved agents for parenteral administration, of which rhodotorulic acid appears most promising. Its preparation is simpler than desferrioxamine and it is twice as potent as desferrioxamine on a weight basis. Secondly, there are iron chelators for oral administration, of which CHA and the isoniazid-pryridoxal complex seem possible candidates at the present. Thirdly, there is the possibility that depot preparations of desferrioxamine or polymeric hydroxamic acids (Winston & Kirchner, 1976) might be of value for parenteral use so that prolonged action can be obtained without the need for continuous infusion.

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Annotation 3 Increasing the Effectiveness .f Iron Chelation

In most experimental and therapeutic situations iron available to exogenous chelating agents is derived from an intracellular ‘chelatable pool’ (White et a l , 1976a; Jacobs, 1977). In some cases where circulating transferrin is fully iron saturated there may be an additional, non-trans- ferrin, form of circulating iron available for chelation (Hershko et a l , 1978a). It is theoretically possible to remove iron from transferrin itself. A number of methods have been proposed to increase the effectiveness of iron chelation, usually with respect to desferrioxamine.

Ascorbic acid has been used for some time as an adjuvant to desferrioxamine therapy. It is presumably effective because it increases the intracellular labile iron pool a t the expense of ferritin iron (Jacobs, 1977). Ferritin iron may also be rapidly mobilized experimentally by dihydroflavins and their analogues (Jones et a l , 1978) and it is possible that such compounds could also increase the amount of chelatable iron. Other manoeuvres designed to improve the delivery of chelators to the target cells by packing them either in liposomes or in resealed erythrocytes have not yet approached the realms of practicality.

A number of other therapeutic approaches remain to be exploited. Hershko et a1 (1978b) have shown that some chelating agents such as DTPA combine primarily with reticuloendoth- elial iron while others, such as CHA, are active mainly in the hepatocyte. The reasons for this are not clear but multiple therapy directed a t different sites is a theoretical possibility. Schubert & Derr (1978) carried out an elegant study of plutonium and cadmium poisoning in mice, showing that mixed ligand chelate therapy is far more effective than the use of a single chelator in reducing the body load of metal even when one of the chelators is largely inactive by itself.

There is some attraction in the possibility of therapeutic removal of iron from transferrin, though so far this can only be effected by agents whose toxicity remains in doubt. Not only is transferrin an ever-present source of iron but when it is depleted of metal it acts as an extracellular trap for iron within cells. Pollack et a1 (1976) have shown that while the time taken for transferrin iron to be mobilized by desferrioxamine is far too long for a therapeutic effect, transfer of the metal can be facilitated by the presence of an intermediate complexing agent, such as citrate or nitriloacetate which can form ternary complexes with transferrin and iron. It is suggested that this is then followed by the release of the iron complex which is readily available for binding by desferrioxamine.

Problems in Chelation Therapy The primary aim of chelation therapy is the prevention of iron toxicity. Unfortunately there

is little agreement regarding the mechanism of toxicity and the practical objective is often taken to be the prevention or removal of iron overload. In an established case of transfusional overload the body may contain 50-60 g iron which, if it were all available for chelation and if chelation were 100% efficient, would require the administration of over half a kilogram of desferrioxamine. The monitoring of such iron overload is possible either by serial liver biopsy or estimation of serum ferritin (Letsky et al, 1974). The definition of toxicity and its clinical assessment is more difficult. At a cellular level it is not certain whether haemosiderin itself causes damage, whether the labile iron pool is important, or whether lysosomal damage is the essential feature. However, clinical signs of toxicity may be influenced by chelation therapy, even when large iron stores remain, and this suggests that the immediate target of chelation therapy is the labile pool of iron within the cells. Clinical toxicity can be monitored by simple

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4 Annotation

growth measurements and evaluation of endocrine status (Modell, 1977), evaluation of hepatic fibrosis by liver biopsy (Risdon et al, 1975) or monitoring cardiac function by echo-cardio- graphy (Henry et al, 1978). It has generally been held that fibrosis in affected organs is a consequence of cell damage but Hunt et a l ( l979) have shown that desferrioxamine has a direct inhibitory effect on collagen synthesis, presumably through neutralization of the matabolically active iron pool necessary for this process. Cells showing large lysosomal iron accumulations do not necessarily show signs of iron toxicity (Jacobs et al, 1978). It is possible that chelation therapy that continuously neutralized a toxic iron pool would be clinically effective without necessarily reducing overall storage levels.

A number of problems need to be resolved before the optimal iron chelation treatment is found. The ideal chelator for clinical use would be cheap to produce, non-toxic, highly efficient in binding iron with complete excretion of the resulting complex. It would be effective when given by mouth, though administration by this route implies some loss of effectiveness because ofchelation of food iron in the gut before absorption. Drugs cheaper than desferrioxamine should not be difficult to devise, and CHA may be an example, but each new drug requires detailed toxicity testing before its introduction for routine use. For this reason alone, desferrioxamine has a considerable practical advantage, though even in this case the long-term toxicity is not known. While it is known to inhibit a large number of iron dependent enzyme systems, such as those involved with DNA synthesis, this is usually only the case at concentrations above the therapeutic range. All effective iron chelators will inhibit iron dependent enzymes to some degree, probably related to their efficiency as chelators. The wide range of such vulnerable metabolic points should be appreciated by investigators (Jacobs &. Worwood, 1974). A specific point of potential vulnerability for patients with disordered erythropoiesis is the possible entry of the chelator into the erythroblast and interference with haem synthesis. High efficiency of a chelator depends on stoichiometric reaction between the compound and available iron. In the case of desferrioxamine the major factor reducing efficiency to levels as low as 10% is the instability of the drug in the body and its rapid excretion (Summers et al, 1979). Despite all the potential problems the present active interest in this area gives hope that further improvements in therapeutic techniques may be possible.

ALLAN JACOBS

Department of Haematology, Welsh National School of Medicine, Heath Park, CardiflCF4 4XW

REFERENCES

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GRADY, R.W., GRAZIANO, J.H., AKERS, H.A. & CERAMI, A. (1976) The development of new iron- chelating drugs. Journal of Pharmacology and Experi- mental Therapeutics, 196, 478-485.

GRADY, R.W., GRAZIANO, J.H., W H ~ E , G.P., JACOBS, A. & CERAMI, A. (1978) The development of new

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GRAZIANO, J.H., GRADY, R.W. & CERAMI, A. (1974) The identification of 2,3-dihydroxybenzoic acid as a potentially useful iron-chelating drug. Journal of Pharmacology and Experimental Therapeutics, 190, 570-575.

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Page 5: Annotation IRON CHELATION THERAPY FOR IRON LOADED PATIENTS

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