Selection of a new generation of orally active α-ketohydroxypyridine iron chelators intended for use in the treatment of iron overload

The prospect of selecting oral α-ketohydroxypyridine chelators intended for clinical use in iron overload has been examined using several animal models of efficacy and toxicity. Studies using iron dextran-loaded mice labelled with ⁵⁹Fe have shown that only the 1-substituted methyl, ethyl, (n)propyl, allyl, cyclopropyl, 2′-methoxyethyl, 3′-ethoxypropyl, or 2-methyl- or 2-ethyl- 3-hydroxypyrid-4-one chelators were orally effective in increasing iron (⁵⁹Fe) excretion by comparison to intraperitoneally administered desferrioxamine at the same dose (250 mg/kg). In contrast, chelators containing -H, mono- or dihydroxyalkyl and diethoxyethyl 1-substituents caused very little or no increase in iron (⁵⁹Fe) excretion by the oral or intraperitoneal routes. In vitro studies using ferritin and haemosiderin have shown that equivalent iron release took place with both groups of chelators irrespective of their in vivo effects. In most cases there was no correlation between the n-octanol/water partition coefficient (K_par) and iron removal efficacy but positive correlation between the lipophilicity and acute or subacute toxicity of these chelators in rats. The most toxic chelator in the chronic toxicity studies in rats was the lipophilic 1, 2-diethyl-3-hydroxypyrid-4-one (EL1NEt). The most effective chelator in increasing iron excretion in mice and rabbits was 1-allyl-2-methyl-3-hydroxypyrid-4-one (L1NAll), and the chelator with the highest safety margin in mice and rats was 1, 2-dimethyl-3-hydroxypyrid-4-one (L1). Overall the oral effectiveness in increasing iron excretion by these chelators in animals does not appear to be related to their lipophilicity or their ability to mobilise polynuclear iron in vitro but rather to other properties possibly related to their rate of biotransformation and excretion. © 1993 Wiley-Liss, Inc.     

Origin and fate of iron mobilized by the 3-hydroxypyridin-4-one oral chelators: studies in hypertransfused rats by selective radioiron probes of reticuloendothelial and hepatocellular iron stores.

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.     

Desferrithiocin is an effective iron chelator in vivo and in vitro but ferrithiocin is toxic

The efficacy and toxicity of the siderophore desferrithiocin (DFT), which has shown potential application in iron chelation therapy, were assessed in vivo and in vitro. DFT was evaluated in vivo in two ways: firstly, by measuring the effect of a single dose of DFT (10-100 mg/kg) on 59Fe excretion in iron-loaded rats labelled with 59Fe; and secondly, by examining the effect of the daily oral administration for 2 weeks of DFT (10-25 mg/kg/d) on the growing rat. DFT and its ferric complex, ferrithiocin (FT), were assessed in vitro from their effects on transferrin and iron uptake and mobilization from rat hepatocytes in culture using transferrin doubly labelled with 125I and 59Fe. Both oral and subcutaneous DFT were highly effective in promoting iron excretion in vivo, but showed evidence of toxicity after oral administration for 2 weeks at 25 mg/kg/d. In addition, DFT was much more effective than desferrioxamine or pyridoxal isonicotinyl hydrazone in reducing hepatocyte iron in vitro. However, FT was cytotoxic, causing membrane disruption and release of intracellular aspartate aminotransferase. It was concluded that DFT should not be considered for chronic iron chelation therapy without extensive further evaluation.     

Studies of an oral iron chelator: 1,2 -dimethyl- 3 -hydroxy-pyrid-4-one I. IRON EXCRETION IN RATS: DEVELOPMENT OF A NEW RAPID MICROWAVE METHOD FOR IRON ANALYSIS IN FAECES

We have developed a simple, rapid method for analysing faecal iron using a microwave oven for digestion followed by atomic absorption spectrometry. The chelating effect of 1,2-dimethyl-3-hydroxy-pyrid-4-one (DMHP) has been tested in rats with experimental iron overloading and three routes of DHMP administration, oral, subcutaneous, and intraperitoneal, have been compared. Regardless of the route of administration, we have found that DMHP promotes iron excretion via the urine. We have not observed a difference in the amount of iron excreted in the faeces before and after DMHP administration by any route. The subcutaneous route of administration is the most effective in promoting iron excretion, followed by the intraperitoneal route. Although most convenient for clinical use, oral administration promotes the excretion of only a small fraction of that by the subcutaneous route.     

Selection of hydroxypyridin-4-ones for the treatment of iron overload using in vitro and in vivo models

The hydroxypyridin-4-one group of iron chelators show promise as potential compounds for the treatment of iron overload by the oral route. In the search for the compounds best suited for long term clinical use, a balance has to be struck between the desire to mobilise the maximum amount of iron and the wish to minimise the potential toxicity of such compounds. In this article we review the approach we have used to evaluate which of the hydroxypyridinones have the properties best suited for further development prior to clinical trials in man. The diversity of a number of closely related compounds substituted on the ring nitrogen have allowed us to study the properties of chelators responsible for cellular mobilisation of iron(III), as well as those which may contribute to their toxicity. The primary hepatocyte culture model has facilitated the investigation of the contribution of their iron binding constant, as well as the critical importance of their relative lipid solubility to both cellular iron mobilisation and toxicity. Similarly studies in mice have confirmed that the factors affecting cellular iron release also control iron excretion in whole animals. Further we have demonstrated that the acute toxicity of this group of compounds is closely linked to the size of the available iron pool.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution of desferrioxamine and hydroxypyridin-4-one chelators in K562 cells affects chelation of intracellular iron pools

Summary The interactions of iron chelators with intracellular iron pools have been examined by measuring the subcellular distribution of radiolabelled desferrioxamine (DFO) and the orally active hydroxypyridinone (HPO) chelator 1, 2-diethyl-3-hydroxypyridin-4-one (CP94), as well as the ability of these chelators to modify the subcellular distribution of ⁵⁹Fe delivered by the receptor mediated endocytosis of transferrin. K562 cells were pulsed with ⁵⁹Fe transferrin and challenged with DFO or CP94 (100 μm IBE) for 20 or 240 min and then subjected to subcellular fractionation. At 20 min there was a significant decrease (P <0.45) in both lysosomal/particulate ⁵⁹Fe (75% of control) and cytosolic ⁵⁹Fe ferritin (50% of control) in cells incubated with CP94, unlike cells treated with DFO where no decrease was observed. By 240 min, in addition to the above, ⁵⁹Fe accumulation was significantly decreased in the nuclear, mitochondrial, and low molecular weight cytosolic fractions with CP94 (P < 0.05). With DFO a significant decrease in ⁵⁹Fe in only the lysosomal/particulate and cytosolic ferritin compartments was observed at 240 min (P <045). At this time, however, there was a significant accumulation of both cytosolic low molecular weight ⁵⁹Fe and cytosolic DFO. The relatively rapid decrease of ⁵⁹Fe within intracellular compartments seen with CP94 compared to DFO was paralleled by a significantly higher accumulation of CP94 than DFO in nuclear, lysosomal/particulate and low molecular weight cytosolic compartments at 20 min (P <0 05). These results suggest that transferrin derived endosomal iron may be chelated by HPOs, unlike DFO, due to their faster uptake into these organelles. The more rapid access of HPOs than DFO to certain intracellular iron pools may explain the greater possibility of HPOs to inhibit proliferation of cells in vivo.     

Comparison of the subacute toxicity and efficacy of 3-hydroxypyridin-4- one iron chelators in overloaded and nonoverloaded mice

Five orally effective iron chelators of the 3-hydroxypyridin-4-one series have been administered intraperitoneally to iron-overloaded and nonoverloaded male mice at a dose of 200 mg/kg/24 h for a total of 60 days to investigate the effect on iron loading and toxicity. There was a significant reduction in hepatic iron at the end of the study in the iron-overloaded mice with all compounds studied using chemical iron quantitation (P less than .001) and with Perls' stain (P less than .01). Liver iron removal with the hydroxypyridinones ranged from 37% with CP20 to 63% with CP51, compared with 46% removal for desferrioxamine (DFO). There was no significant reduction in splenic or cardiac iron with any chelator. There were no deaths in iron-overloaded animals receiving any of the hydroxypyridin-4-ones, but significantly more deaths in the nonoverloaded groups as a whole (P less than .03). No weight loss was observed with any chelator. Significant reductions in hemoglobin and white cell count were observed with CP20(L1). No histologic abnormalities of kidney, spleen, bone marrow, or stifle joints were observed. Intracytoplasmic inclusion bodies were observed in the centrilobular hepatocytes of animals administered each of the hydroxypyridin-4-ones, while the DFO-treated and control groups showed no such changes.     

Biliary Iron Excretion in Rats following Pyridoxal Isonicotinoyl Hydrazone

S ummary Biliary excretion of iron after administration of pyridoxal isonicotinoyl hydrazone (PIH), a recently identified effective iron-chelating agent, was investigated in rats. PIH administered both intraperitoneally and orally was shown to increase significantly ⁵⁹Fe excretion into bile of rats which had previously been injected with ⁵⁹Fe-transferrin to label hepatic parenchymal cells. ⁵⁹Fe-PIH appears in bile as early as 15 min after chelator administration and the peak of ⁵⁹Fe-radioactivity in bile is seen 1–5 h following intraperitoneal PIH injection. PIH, administered intraperitoneally, 125–250 mg/kg, increased 24 h biliary radioiron excretion about 35 times and in addition increased urinary and faecal iron excretion. When PIH was given immediately before ⁵⁹Fe-transferrin, 24 h cumulative biliary ⁵⁹Fe excretion was even higher. PIH was also demonstrated to increase biliary excretion of radioiron released from ⁵⁹Fe-haemoglobin catabolysed in reticuloendothelial cells. The effect of PIH was confirmed by estimation of biliary iron concentration using the method of atomic absorption spectrophotometry. Repeated PIH administration to rats decreased ⁵⁹Fe radioactivity in liver and kidney and increased urinary and faecal iron excretion.     

A rapid assay for evaluation of iron-chelating agents in rats

The animal assay of potential new iron-chelating agents is at present dependent on cumbersome and imprecise iron balance studies in hypertransfused rodents. We report the development of a radioisotope assay in intact rats based on the transient labeling by ferritin 59Fe of the main source of chelatable iron within hepatocytes. The isotope was maximally available to chelators during the first 6 hr after its injection, nearly all the excretion being in the bile. The bile 59Fe/total iron ratio was independent of both the chelator and its dose. However, in iron-loaded rats, the ratio was reduced, and the isotope excretion was a less sensitive measure of intrahepatic chelation. In the proposed assay, test chelators were given to normal rats 2 hr after an intravenous injection of 59Fe-ferritin. Four hours later, the radioiron in the liver and in the gut gave a sensitive measure of the mobilization of hepatic iron to the bile. In addition, chemical iron determinations identified a small alternative source of urinary chelate with agents known to promote urine excretion in man. The assay gave a rapid and precise screen for chelators given by parenteral and oral routes.     

Iron chelation therapy

Although iron chelation therapy with deferoxamine (DFO) has changed life expectancy in thalassemic patients, compliance with the rigorous requirements of long-term subcutaneous DFO infusions is unsatisfactory. This problem underlines the current efforts for developing alternative, orally effective chelators to improve compliance and treatment results. For the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available. The most important of the new generation of oral chelators are deferiprone and ICL670. Total iron excretion with deferiprone is less than with DFO, but deferiprone has a better ability to penetrate cell membranes and may have a better cardioprotective effect than DFO. Current studies of the clinical efficacy and tolerability of ICL670 indicate that at a single oral dose of 20 mg/kg daily, it may be as effective as parenteral DFO used at the standard dose of 40 mg/kg daily. Combined chelation treatment, employing a weak chelator that penetrates cells better, and a stronger chelator with efficient urinary excretion, may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.     

Dose response studies using desferrioxamine and orally active chelators in a mouse model

59Fe excretion studies in response to different doses (4-9 mg) of three N-substituted 3-hydroxypyrid-4-one chelators, (1,2-dimethyl-3-hydroxypyrid-4-one, 1-ethyl-2-methyl-3-hydroxypyrid-4-one, 1-propyl-2-methyl-3-hydroxypyrid-4-one), and desferrioxamine in iron overloaded 59Fe lactoferrin labelled mice (40 +/- 4 g) have shown that the former chelators, when administered intraperitoneally and intragastrically, caused comparable 59Fe excretions to intraperitoneal desferrioxamine of equivalent doses. No apparent ill effects were observed when doses of 300 mg/kg were administered for 24 d.     

PCTH: a novel orally active chelator for the treatment of iron overload disease

Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of this series of chelators, both in cell culture and in animal models. Several PCIH analogs were shown to be effective at inducing iron mobilization and preventing iron uptake from the iron-transport protein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrion and inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orally active and well tolerated by mice at doses ranging from 50 to 100 mg kg(-1), twice daily (b.d.). A dose-dependent increase in fecal 59Fe excretion was observed in the PCTH-treated group. This level of iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for the treatment of beta-thalassemia (thal) and Friedreich's Ataxia (FA).     

PCTH: A Novel Orally Active Chelator for the Treatment of Iron Overload Disease

Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of this series of chelators, both in cell culture and in animal models. Several PCIH analogs were shown to be effective at inducing iron mobilization and preventing iron uptake from the iron-transport protein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrion and inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orally active and well tolerated by mice at doses ranging from 50 to 100 mg kg^?1, twice daily (b.d.). A dose-dependent increase in fecal ⁵⁹Fe excretion was observed in the PCTH-treated group. This level of iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for the treatment of β-thalassemia (thal) and Friedreich's Ataxia (FA).     

Oral iron chelators--development and application

Iron chelation therapy is the only therapeutic approach that leads to enhanced iron excretion in beta-thalassaemia major and other transfusion-dependent patients. Although desferrioxamine has been used in such treatment over the last three decades, it is not an ideal drug due to its poor oral availability. Consequently extensive research effort has been directed towards the identification of non-toxic, orally active iron chelators. An ideal candidate must possess a range of critical physicochemical and biological properties, such as high selectivity and affinity for iron(III), tightly controlled distribution and metabolic profiles and low toxicity. Unfortunately, hexadentate ligands are generally associated with poor oral bioavailability, whereas many tridentate and bidentate molecules are orally active. The tridentate triazoles have been investigated for clinical potential; they are readily absorbed from the gastrointestinal tract and promote iron excretion with high efficacy. In similar fashion, several bidentate hydroxypyridinones have been demonstrated to possess potential as oral chelating agents.     

Iron Absorption in Patients with Chronic Uraemia

The purpose of this study was to examine the accuracy of iron absorption calculated from the incorporation of radioiron into red cells (RCI), compared to measurement by the whole body counting technique (WBR). RCI of orally administered ⁵⁹Fe, and absorption of ⁵⁹Fe assessed by WBR were measured simultaneously in 53 chronic uraemic patients (16 non-dialysed, 18 peritoneal dialysed, 19 haemodialysed), 14 renal transplanted patients with normal renal function, and 27 healthy subjects. In the majority of subjects RCI values were lower than corresponding WBR values, with mean red cell ⁵⁹Fe utilization values (RCI/WBR ratio x 100) in the various groups from 78% to 93%. All groups demonstrated significant correlations between RCI and WBR with r values from 0.963 to 0.996 (P<0.001). RCI was higher in patients with reduced marrow iron stores than in patients with adequate iron stores (P<0.001), and was correlated both to plasma transferrin (r = 0.59, P<0.001) and serum ferritin (r = -0.88, P <0.001). In all groups there was good accuracy of calculated iron absorption (from RCI) compared to ‘true’ iron absorption (by WBR) with r values from 0.963 to 0.995 (P <0.001). Iron absorption measurement based on red cell incorporation appears to be a practical and accurate alternative to whole body counting, both in healthy subjects and in patients with chronic uraemia.     

Liposome Entrapped Desferrioxamine and Iron Transporting Ionophores: a New Approach to Iron Chelation Therapy

Liposome-entrapped desferrioxamine was administered to iron-over-loaded 59Fe lavelled mice. When given orally or intraperitoneally entrapment did not enhance the effect of the chelator, but given intravenously liposomal desferrioxamine doubled the 59Fe excretion for a given dose of the drug, and excretion after a single dose continued for up to 3 d. In addition, liposomes containing ionophore A23187 administered concurrently with DTPA caused an excretion of 59Fe, whereas DTPA alone had no effect.     

Excretion patterns of alkylating metabolites in urine following cyclophosphamide treatment of tumor patients: Influence of application route,dosage, liver and kidney function

The excretion patterns of cyclophosphamide (CP) in urine were studied in 54 tumor patients aged between 21 and 61 years, using the nitrobenzyl-pyridine (NBP) reaction, with regard to the route of application (i.v., i.m. or oral), the CP dose and the functional state of the liver and kidney. The studies were carried out in nephrectomized patients and patients with liver affections caused by the basic disease, in particular with malignant lymphomas and mammary carcinomas. The following results were obtained: 1. There exists a direct relationship between the dose of CP applied and the quantitative excretion of alkylating metabolites in urine. According to these studies in which the patients received up to 2.8 g CP/m2 body surface, the upper CP dose was limited by the generally toxic side effects rather than by the metabolization rate. 2. At comparable CP doses the route of application (i.v., i.m. or oral) has no appreciable influence on the excreted NBP activity. 3. Disorders of the liver function without signs of icterus are not a contraindication to CP treatment. 4. The functional failure of one kidney has no statistically significant influence on the excretion of alkylating metabolites in urine.     

Action of chelators in iron-loaded cardiac cells: Accessibility to intracellular labile iron and functional consequences

Labile iron in hemosiderotic plasma and tissue are sources of iron toxicity. We compared the iron chelators deferoxamine, deferiprone, and deferasirox as scavengers of labile iron in plasma and cardiomyocytes at therapeutic concentrations. This comprised chelation of labile plasma iron (LPI) in samples from thalassemia patients; extraction of total cellular iron; accessing labile iron accumulated in organelles and preventing formation of reactive-oxidant species; and restoring impaired cardiac contractility. Neonatal rat cardiomyocytes were used for monitoring chelator extraction of LCI (labile cell iron) as 59Fe; assessing in situ cell iron chelation by epifluorescence microscope imaging using novel fluorescent sensors for iron and reactive oxygen species (ROS) selectively targeted to organelles, and monitoring contractility by time-lapse microscopy. At plasma concentrations attained therapeutically, all 3 chelators eliminated LPI but the orally active chelators rapidly gained access to the LCI pools of cardiomyocytes, bound labile iron, attenuated ROS formation, extracted accumulated iron, and restored contractility impaired by iron overload. The effect of deferoxamine at therapeutically relevant concentrations was primarily by elimination of LPI. The rapid accessibility of the oral chelators deferasirox and deferiprone to intracellular labile iron compartments renders them potentially efficacious for protection from and possibly reversal of cardiac damage induced by iron overload.     

HBED ligand: preclinical studies of a potential alternative to deferoxamine for treatment of chronic iron overload and acute iron poisoning

We have continued the preclinical evaluation of the efficacy and safety of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl) ethylenediamine-N, N'-diacetic acid monosodium salt (NaHBED) for the treatment of both chronic transfusional iron overload and acute iron poisoning. We examined the effect of route of administration by giving equimolar amounts of NaHBED and deferoxamine (DFO) to Cebus apella monkeys as either a subcutaneous (SC) bolus or a 20-minute intravenous (IV) infusion. By both routes, NaHBED was consistently about twice as efficient as DFO in producing iron excretion. For both chelators at a dose of 150 micromol/kg, SC was more efficient than IV administration. The biochemical and histopathologic effects of NaHBED administration were assessed. No systemic toxicity was found after either IV administration once daily for 14 days to iron-loaded dogs or after SC administration every other day for 14 days to dogs without iron overload. Evidence of local irritation was found at some SC injection sites. When the NaHBED concentration was reduced to 15% or less in a volume comparable to a clinically useful one, no local irritation was found with SC administration in rats. Because treatment of acute iron poisoning may require rapid chelator infusion, we compared the effects of IV bolus administration of the compounds to normotensive rats. Administration of DFO produced a prompt, prolonged drop in blood pressure and acceleration of heart rate; NaHBED had little effect. NaHBED may provide an alternative to DFO for the treatment of both chronic transfusional iron overload and of acute iron poisoning.     

Iron mobilization from myocardial cells by 3-hydroxypyridin-4-one chelators: studies in rat heart cells in culture

The ability of 3-hydroxypyridin-4-ones (CP), a family of bidentate orally effective iron chelators, to remove iron and to prevent iron-induced lipid peroxidation was studied in beating rat myocardial cells in culture. The iron (III) binding constant (log beta 3) of all CP compounds is 36, but their lipophilicity may be modified by altering the length of the R2 substituent on the ring nitrogen. There was a direct relation between lipid solubility and chelating efficiency. Although at high concentrations all CP compounds were more effective in iron mobilization than deferoxamine, the opposite was true for low concentrations. Further studies with 1,2-diethyl-3-hydroxypyridin-4-one (CP94), the most effective CP compound, have shown that iron mobilization is completed within 6 hours, that effective mobilization requires a drug: iron molar ratio exceeding 3:1 permitting the formation of a hexadentate complex, and that the beneficial effects of iron mobilization are manifested in a marked reduction in membrane lipid peroxidation as indicated by cellular malonaldehyde content. Our study represents the first demonstration of a direct interaction between myocardial cells and an orally effective iron chelator, and underlines the need for high molar concentrations for achieving an optimal therapeutic effect.