Effective inhibition of copper-catalyzed production of hydroxyl radicals by deferiprone.

Copper ions can catalyze the production of free oxygen radicals (•OH and •OOH) similar to iron ions. The capacity to initiate oxidative damage is most commonly attributed to Cu-induced toxicity in copper-related diseases where there is an increase in copper levels and also when Cu homeostasis and regulation are disrupted. An antioxidant/chelator inhibiting Cu-induced oxidative damage could play a significant role in the treatment of such Cu-related diseases. Deferiprone has high affinity for copper binding and can be considered for the potential treatment of copper toxicity and overloading conditions, such as Wilson's disease. In the present study, the ability of deferiprone to inhibit the production of hydroxyl radicals catalyzed by copper ions was elucidated using an Electron Paramagnetic Resonance (EPR) spin trapping technique. The values of g-factors and hyperfine splitting constants were calculated for Cu(II)-deferiprone 1:1 complex: (a = 58.5 G, g = 2.1667) and 1:2 complex: (a = 73.0 G, g = 2.1378). The TMIO spin trap (2,2,4-trimethyl-2H-imidazole-1-oxide) was used for the detection of free radicals formed in Fenton-like copper-catalyzed reactions. It was demonstrated that the interaction of deferiprone with Cu2+ ions completely inhibited hydroxyl radical (•OH) production in the presence of hydrogen peroxide. It was found also that deferiprone inhibits Cu-induced oxidation of linoleic acid in micellar solution. In addition to existing data for water solutions, the affinity of deferiprone for copper binding in non-aqueous environment has been elucidated.

Inhibition of Fe(2+)- and Fe(3+)- induced hydroxyl radical production by the iron-chelating drug deferiprone.

Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe(2+) and Fe(3+) ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe(2+) have reported ferroxidase activity by L1 followed by the formation of Fe(3+) chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1-Fe(3+) complexes exhibit intense absorption bands up to 800nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe(2+) and Fe(3+) ions and (2) does UV-Vis irradiation of the L1-Fe(3+) complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe(3+) aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO-OH, a(N)=14.15G, a(H)=16.25G; PBN-OH, a(N)=16.0G, a(H)=2.7G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe(2+) ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage.

Safety issues of iron chelation therapy in patients with normal range iron stores including thalassaemia, neurodegenerative, renal and infectious diseases.

An increased number of thalassaemia patients treated with effective chelation therapy protocols are achieving body iron levels similar to those of normal individuals. Iron chelation therapy has also been recently used in a number of other categories of patients with no excess body iron load such as neurodegenerative, renal and infectious diseases. Chelation therapy in the absence of iron overload in the latter conditions raises many safety issues including chelator overdose toxicity and toxicity related to iron and other essential metal deficiencies. Preliminary preclinical and clinical toxicity evidence suggest that deferoxamine and deferasirox can only be safely used for these non-iron loaded conditions for short-term treatments of a few weeks, whereas deferiprone can be used for longer term treatments of many months. The selection of the chelating drug and appropriate dose protocols for targeting specific organs and conditions is critical for the safety of patients with normal iron stores. Chelation therapy is likely to play a major role as adjuvant, alternative or main therapy in many non-iron loading conditions in the forthcoming years.

The design and development of deferiprone (L1) and other iron chelators for clinical use: targeting methods and application prospects.

Iron is essential for all human cells as well as neoplastic cells and invading microbes. Natural and synthetic iron chelators could affect biological processes involving iron and other metal ions in health and disease states. Iron overload is the most common metal toxicity condition worldwide. There are currently two iron chelating drugs, which are mostly used for the treatment of thalassaemia and other conditions of transfusional iron overload. Deferoxamine was until recently the only approved iron chelating drug, which is effective but very expensive and administered parenterally resulting in low compliance. Deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) is the world's first and only orally active iron chelating drug, which is effective and inexpensive to synthesise thus increasing the prospects of making it available to most thalassaemia patients in third world countries who are not currently receiving any form of chelation therapy. Deferiprone has equivalent iron removal efficacy and comparable toxicity to deferoxamine. There are at least four other known iron chelators, which are currently being developed. Even if successful, these are not expected to become available for clinical use in the next five years and to be as inexpensive as deferiprone. The variation in the chemical, biological, pharmacological, toxicological and other properties of the chelating drugs and experimental chelators provide evidence of the difference in the mode of action of chelators and the need to identify and select molecular structures and substituents based on structure/activity correlations for specific pharmacological activity. Such information may increase the prospects of designing new chelating drugs, which could be targeted and act on different tissues, organs, proteins and iron pools that play important role not only in the treatment of iron overload but also in other diseases of iron and other metal imbalace and toxicity including free radical damage. Chelating drugs could also be designed, which could modify the enzymatic activity of iron and other metal containing enzymes, some of which play a key role in many diseases such as cancer, inflammation and atherosclerosis. Other applications of iron chelating drugs could involve the detoxification of toxic metals with similar metabolic pathways to iron such as Al, Cu, Ga, In, U and Pu.

Molecular factors affecting the complex formation between deferiprone (L1) and Cu(II). Possible implications on efficacy and toxicity.

Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, L1, CAS 30652-11-0) is a new chelating drug used worldwide for the treatment of iron overloading conditions. Spectrophotometric and potentiometric measurements were carried out to investigate the interaction of L1 with Cu(II) ions under different conditions. The complexation of Cu(II) ions with L1 in aqueous solution leads predominantly to the formation of the Cu(L1)2 species at a pH range of 4-9. The experimental results indicate that L1 has high affinity for Cu(II) with stability constants log beta 11 = 10.3 +/- 0.9 and log beta 12 = 19.2 +/- 0.6. The effect of Cu(II) ions on the affinity of L1 for Fe(III) ions by competition reactions in vitro indicate displacement of Fe(III) in a concentration dependent manner by Cu(II). Similarly, the presence of different buffers at various pH values resulted in the formation of different stoichiometry L1 complexes with Cu(II) and of mixed complexes with buffer anions. The strong interaction of L1 with Cu(II) may have implications on the therapeutic and toxicological properties of this chelating drug. In particular, L1 may be used in the treatment of copper overloading conditions, such as Wilson's disease or other conditions where copper toxicity is implicated.

Transfusional iron overload and chelation therapy with deferoxamine and deferiprone (L1).

Iron is essential for all living organisms. Under normal conditions there is no regulatory and rapid iron excretion in humans and body iron levels are mainly regulated from the absorption of iron from the gut. Regular blood transfusions in thalassaemia and other chronic refractory anaemias can result in excessive iron deposition in tissues and organs. This excess iron is toxic, resulting in tissue and organ damage and unless it is removed it can be fatal to those chronically transfused. Iron removal in transfusional iron overload is achieved using chelation therapy with the chelating drugs deferoxamine (DF) and deferiprone (L1). Effective chelation therapy in chronically transfused patients can only be achieved if iron chelators can remove sufficient amounts of iron, equivalent to those accumulated in the body from transfusions, maintaining body iron load at a non-toxic level. In order to maintain a negative iron balance, both chelating drugs have to be administered almost daily and at high doses. This form of administration also requires that a chelator has low toxicity, good compliance and low cost. DF has been a life-saving drug for thousands of patients in the last 40 years. It is mostly administered by subcutaneous infusion (40-60 mg/kg, 8-12 h, 5 days per week), is effective in iron removal and has low toxicity. However, less than 10% of the patients requiring iron chelation therapy worldwide are able to receive DF because of its high cost, low compliance and in some cases toxicity. In the last 10 years we have witnessed the emergence of oral chelation therapy, which could potentially change the prognosis of all transfusional iron-loaded patients. The only clinically available oral iron chelator is L1, which has so far been taken by over 6000 patients worldwide, in some cases daily for over 10 years, with very promising results. L1 was able to bring patients to a negative iron balance at doses of 50-120 mg/kg/day. It increases urinary iron excretion, decreases serum ferritin levels and reduces liver iron in the majority of chronically transfused iron-loaded patients. Despite earlier concerns of possible increased risk of toxicity, all the toxic side effects of L1 are currently considered reversible, controllable and manageable. These include agranulocytosis (0.6%), musculoskeletal and joint pains (15%), gastrointestinal complaints (6%) and zinc deficiency (1%). The incidence of these toxic side effects could in general be reduced by using lower doses of L1 or combination therapy with DF. Combination therapy could also benefit patients experiencing toxicity with DF and those not responding to either chelator alone. The overall efficacy and toxicity of L1 is comparable to that of DF in both animals and humans. Despite the steady progress in iron chelation therapy with DF and L1, further investigations are required for optimising their use in patients by selecting improved dose protocols, by minimising their toxicity and by identifying new applications in other diseases of iron imbalance.

Comparative efficacy and toxicity of desferrioxamine, deferiprone and other iron and aluminium chelating drugs.

The efficacy and toxicity aspects of the iron and aluminium chelating drugs desferrioxamine and deferiprone (L1, 1,2-dimethyl-3-hydroxypyrid-4-one), have been compared. Major emphasis was given in the use of these two and also of other chelators in conditions of iron overload, imbalance and toxicity, as well as the incidence and possible causes of toxic side effects in both animals and humans. The chemical basis of chelation and the interaction of these chelators with the iron pools are discussed within the context of clinical application in iron overload and other conditions such as renal dialysis, rheumatoid arthritis, cancer, heart disease, malaria, etc. The design and development of new orally active alpha-ketohydroxypyridine and other chelators are considered and compared with 14 other chelators which have been previously tested in man for the removal of iron, most of which, however, were later abandoned because of low efficacy or major toxicity. The design of new therapeutic protocols based on the pharmacological, toxicological and metabolic transformation properties of the chelating drugs is also being considered, within the context of maximising their efficacy and minimising their toxicity. Overall, oral deferiprone appears to be as effective as s.c. desferrioxamine in the removal of iron and aluminium in man and to have a similar but different toxicity profile from desferrioxamine in both animals and man. The low cost and oral activity of deferiprone will make it the drug of choice for the vast majority of patients, who are not currently being chelated either because they cannot afford the high cost of desferrioxamine therapy or are not complying or have toxic side effects with its s.c. administration.

Iron: mammalian defense systems, mechanisms of disease, and chelation therapy approaches.

During the past 6 decades, much attention has been devoted to understanding the uses, metabolism and hazards of iron in living systems. A great variety of heme and non-heme iron-containing enzymes have been characterized in nearly all forms of life. The existence of both ferrous and ferric ions in low- and high-spin configuration, as well as the ability of the metal to function over a wide range of redox potentials, contributes to its unique versatility. Not surprisingly, the singular attributes of iron that permit it to be so useful to life likewise render the metal dangerous to manipulate and to sequester. All vertebrate animals are prone to tissue damage from exposure to excess iron. In order to protect them from this threat, a complex system has evolved to contain and detoxify this metal. This is known as the iron withholding defense system, which mainly serves to scavenge toxic quantities of iron and also for depriving microbial and neoplastic invaders of iron essential for their growth. Since 1970, medical scientists have become increasingly aware of the problems involved in cellular iron homeostasis and of the disease states related to its malfunctioning. Scores of studies have reported that excessive iron in specific tissue sites is associated with development of infection, neoplasia, cardiomyopathy, arthropathy and a variety of endocrine and neurologic deficits. Accordingly, several research groups have attempted to develop chemical agents that might prevent and even eliminate deposits of excess iron. A few of these drugs now are in clinical use, e.g. deferiprone (L1). In the present review, we focus on recent developments in (i) selected aspects of the iron withholding defense system, and (ii) pharmacologic methods that can assist the iron-burdened patient.

New concepts of iron and aluminium chelation therapy with oral L1 (deferiprone) and other chelators. A review.

The introduction of oral chelation therapy with the alpha-ketohydroxypyridine chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, INN/BAN: deferiprone) in iron- and aluminium-overloaded patients has been initiated in over 15 countries in the last 7 years. Over 600 patients with various conditions, in 26 centres have received L1, in some cases daily for over 5 years. In the vast majority of iron-loaded patients, doses of 55-100 mg kg-1 of L1 resulted in urinary iron excretion levels greater than those accumulating from transfusions (15-35 mg d-1) and also reduction in serum ferritin and liver iron to near normal levels. Urinary iron excretion was related to the iron load of the patients, as well as the dose and frequency of administration of L1. The L1 appears to mobilize iron mainly from a serum iron pool in excess of transferrin saturation, transferrin-bound iron and tissue iron, mainly but not exclusively from the liver. The order of metal binding by L1 at pH 7.4 is Fe > Cu > A1 > Zn. Aluminium removal from aluminium-loaded renal dialysis patients by L1 was also effective at doses similar to those used for iron-loaded patients. Overall toxic side effects include six cases of reversible agranulocytosis, 0-30% incidence of transient musculoskeletal and joint pains, 0-6% of gastric intolerance and 0-2% zinc deficiency. Deferiprone appears to be as effective as desferrioxamine in iron and aluminium removal and has low toxicity. Its oral efficacy and low cost make it more accessible than desferrioxamine for the vast majority of patients needing iron chelation worldwide. The development of other alpha-ketohydroxypyridines is currently in progress.

Oral iron chelation therapy with deferiprone. Monitoring of biochemical, drug and iron excretion changes.

The oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone, CAS 30652-11-0) has been given daily for 3-11 months to 6 transfusion dependent iron loaded patients (myelodysplasia (MDS) 2, Diamond-Blackfan anaemia 1, thalassaemia intermedia 1, thalassaemia major 2). Daily doses of 3 g, 2 x 2 g and 3 x 2 g were administered for the first 2-7 months. Daily doses of 2 x 3 g were also used for periods up to 4 months. Urine iron excretion following 3 g of L1 was found to be related to the number of previous transfusions but not to serum ferritin or the amount of L1 excreted. In each case 24 h urinary iron excretion in response to 3 g L1 ranged from 5-21 mg in MDS, 13-25 mg in a thalassaemia intermedia and a Diamond-Blackfan patient and 16-110 mg in thalassaemia major patients. Further increases of urinary iron were observed in all the patients when the daily dose was increased. Serum ferritin levels have fluctuated but overall have remained unchanged. Biochemical assessment did not show any major abnormalities ascribed to L1 except from subnormal serum zinc levels in two patients and white blood cell absorbate in another. In a separate study we have compared urinary L1 and iron excretions in 7 transfusional iron loaded patients. In all the cases the concentration of L1 was in excess of iron and higher than the level required for 100% iron binding. There was no other apparent correlation between the concentrations of L1 and iron in the urines studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of aluminium mobilization in renal dialysis patients using the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one.

The oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone, CAS 30652-11-0) has been tested in 11 renal dialysis patients, 10 for aluminium and 1 for iron mobilization. L1 was administered just after the patients were placed on the haemodialyser and blood samples were collected before haemodialysis at 1 h and for some patients at longer intervals. Plasma aluminium levels before treatment ranged from 12 to 264 micrograms/l. A mean increase of 90% was observed within the first hour of oral administration in 6 patients who received a dose of L1 of 40-60 mg/kg. Plasma aluminium levels then progressively decreased after this period. Three patients with plasma aluminium of 30-66 micrograms/l who received a dose of L1 of less than 30 mg/kg had no significant changes in their plasma aluminium. In 2 other cases administration of L1 resulted in an over 30-fold increase of aluminium concentration in the dialysate of a continuous ambulatory peritoneal dialysis patient and of over 3 times the iron concentration in the dialysate of an iron loaded haemodialysis patient. In the last patient HPLC analysis of the dialysate samples obtained from the haemodialyser has shown complete clearance of L1 within 4 h but not of its glucuronide metabolite within 6.5 h of the L1 administration. No toxic side effects were observed in any of the 11 patients who received oral L1. These are the first clinical trials of an oral chelator in renal dialysis patients which suggest that oral L1 and possibly other alpha-ketohydroxypyridine chelators may have a use in the treatment of patients with aluminium overload.