Optimizing therapy for iron overload in the myelodysplastic syndromes: recent developments

The myelodysplastic syndromes (MDS) are characterized by cytopenias and risk of progression to acute myeloid leukaemia (AML). Most MDS patients eventually require transfusion of red blood cells for anaemia, placing them at risk of transfusional iron overload. In β-thalassaemia major, transfusional iron overload leads to organ dysfunction and death; however, with iron chelation therapy, organ function is improved, and survival improved to near normal and correlated with the degree of compliance with chelation. In lower-risk MDS, several nonrandomized studies suggest an adverse effect of iron overload on survival and that lowering iron with chelation may minimize this impact. Emerging data indicate that chelation may improve organ function, particularly hepatic function, and a minority of patients may have improvement in cell counts and decreased transfusion requirements. While guidelines for MDS generally recommend chelation in selected lower-risk patients, data from nonrandomized trials suggest iron overload may impact adversely on the outcome of higher-risk MDS and stem cell transplantation (SCT). This effect may be due to increased transplant-related mortality, infection and AML progression, and preliminary data suggest that lowering iron may be beneficial in this patient group. Other areas of active and future investigation include optimizing the monitoring of iron overload using imaging such as T2* MRI and measures of labile iron and oxidative stress; correlating new methods of measuring iron to clinical outcomes; clarifying the contribution of different cellular and extracellular iron pools to iron toxicity; optimizing chelation by using agents that access the appropriate iron pools to minimize the relevant clinical consequences in individual patients; and incorporating measures of quality of life and co-morbidities into clinical trials of chelation in MDS. It should be noted that chelation is costly and potentially toxic, and in MDS should be initiated after weighing potential risks and benefits for each patient until more definitive data are available. In this review, data on the impact of iron overload in MDS and SCT are discussed; for example, several noncontrolled studies show inferior survival in patients with iron overload in these clinical settings, including an increase in transplant-related mortality and infection risk. Possible mechanisms of iron toxicity include oxidative stress, which can damage cellular components, and the documented impact of lowering iron on organ function with measures such as iron chelation therapy includes an improvement in elevated liver transaminases. Lowering iron also appears to improve survival in both lower-risk MDS and SCT in nonrandomized studies. Selected aspects of iron metabolism, transport, storage and distribution that may be amenable to future intervention and improved removal of iron from important cellular sites are discussed, as are attempts to quantify quality of life and the importance of co-morbidities in measures to treat MDS, including chelation therapy.     

Iron Overload Exacerbates Busulfan-Melphalan Toxicity Through a Pharmacodynamic Interaction in Mice

Purpose

Busulfan-melphalan high-dose chemotherapy followed by autologous stem cell transplantation is an essential consolidation treatment of high-risk neuroblastoma in children. Main treatment limitation is hepatic veno-occlusive disease, the most severe and frequent extra-hematological toxicity. This life threatening toxicity has been related to a drug interaction between busulfan and melphalan which might be increased by prior disturbance of iron homeostasis, i.e. an increased plasma ferritin level.

Methods

We performed an experimental study of busulfan and melphalan pharmacodynamic and pharmacokinetics in iron overloaded mice.

Results

Iron excess dramatically increased the toxicity of melphalan or busulfan melphalan combination in mice but it did not modify the clearance of either busulfan or melphalan. We show that prior busulfan treatment impairs the clearance of melphalan. This clearance alteration was exacerbated in iron overloaded mice demonstrating a pharmacokinetic interaction. Additionally, iron overload increased melphalan toxicity without altering its pharmacokinetics, suggesting a pharmacodynamic interaction between iron and melphalan. Based on iron homeostasis disturbance, we postulated that prior induction of ferritin, through Nrf2 activation after oxidative stress, may be associated with the alteration of melphalan metabolism.

Conclusion

Iron overload increases melphalan and busulfan-melphalan toxicity through a pharmacodynamic interaction and reveals a pharmacokinetic drug interaction between busulfan and melphalan.

     

Protein adducts of malondialdehyde and 4-hydroxynonenal in livers of iron loaded rats: quantitation and localization

Pathophysiological mechanisms for hepatocellular injury, fibrosis and/or cirrhosis in hepatic iron overload are poorly understood. An increase in intracellular transit pool of iron can catalyze peroxidation of lipids to produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Covalent binding of such lipid aldehydes with proteins may cause impairment in cellular function and integrity. This investigation was focused on quantitative determination of MDA and HNE-protein adducts, and to establish a correlation between iron deposition and formation and localization of MDA and HNE-protein adducts, using immunohistochemistry. To achieve iron overload, male SD rats were fed a 2.5% carbonyl iron-supplemented diet for six weeks, while control animals received standard diet. Total iron as well as low molecular weight chelatable iron (LMWC-Fe) in the hepatic tissue of rats fed the iron supplemented diet increased significantly ( approximately 14- and approximately 15-fold, respectively). Quantitative ELISA for MDA-and HNE-protein adducts showed remarkable increases of 186 and 149%, respectively, in the liver homogenates of rats fed the iron-supplemented diet. Sections of liver stained for iron showed striking iron deposits in periportal (zone 1) hepatocytes, which was less dramatic in midzonal (zone 2) cells. Livers from iron-loaded rats showed strong, diffuse staining for both MDA and HNE adducts, which was highly pronounced in centrilobular (zone 3) hepatocytes, but was also evident in midzonal cells (zone 2). The demonstration of greater formation of both MDA and HNE-protein adducts provides evidence of iron-catalyzed lipid peroxidation in vivo. Although in this model of iron overload there was no evidence of tissue injury, our results provide an account of some of the initiating factors or early molecular events in hepatocellular damage that may lead to the pathological manifestations seen in chronic iron overload.     

High hepcidin level accounts for the nigral iron accumulation in acute peripheral iron intoxication rats

Hepcidin is considered to be a circulatory hormone and a major mechanism regulating iron homeostasis. Our previous publication revealed that acute iron intoxication induced iron deposit and dopaminergic neuron degeneration in the substantia nigra (SN) of a rat model. However, whether and how hepcidin functions in this nigral iron accumulation has not been elucidated. In the present study, we observed a decreased of FPN1 protein level in the SN triggered by peripheral iron overload within 4 h, which correlated with a high hepcidin level. To further investigate the role of intracellular hepcidin under iron overload circumstances, we assessed the expression of hepcidin mRNA and FPN1 protein in vitro. We observed that hepcidin mRNA level was up-regulated and FPN1 protein level was down-regulated in MES23.5 dopaminergic cells in a period of 4h incubation with iron. Both in pCMV-XL4-hepcidin transfected and hepcidin-treated cells, decreased FPN1 protein levels were observed. Our data provide direct evidence that the role for intracellular hepcidin generated in the SN is particularly relevant to restrict iron release by down-regulation FPN1 expression in this region, thus an important contributor to the abnormal iron deposit occurred at an early stage in conditions of peripheral iron intoxication.     

铝过负荷致小鼠脑神经元退变与脑铁代谢失衡的关系

目的探讨铝致神经元退行性变是否与脑铁代谢失衡有关。方法每只鼠侧脑室内分别注射0.125%,0.25%和0.5%的Al溶液3μL(AlCl3·6H2O配制,以Al计算浓度),每日1次,连续5d(d1~d5),建立铝过负荷小鼠模型。于d10,d20和d30用跳台法和水迷宫测定小鼠学习记忆能力、海马病理形态学改变、海马单胺氧化酶B(MAO-B)活性、海马线粒体铁蛋白水平和全脑铁水平。结果铝过负荷致小鼠学习记忆能力明显下降,海马CA1区神经元核固缩和神经细胞丢失,脑内铁水平明显升高,海马线粒体内铁蛋白水平明显降低、MAO-B活力显著升高,均呈剂量依赖性和时间依赖性改变。结论铝过负荷致神经元退变可能与其干扰脑铁代谢有关。     

Design,synthesis, and physicochemical and biological characterization of a new iron chelator of the family of hydroxychromenes

Increasing evidence suggests that iron plays an important role in tissue damage both during chronic iron overload diseases (i.e., hemochromatosis) and when, in the absence of actual tissue iron overload, iron is delocalized from specific carriers or intracellular sites (inflammation, neurodegenerative diseases, postischaemic reperfusion, xenobiotic intoxications, etc.). In the present work, we appropriately modified an iron chelator of the hydroxychromene family in order to obtain a tridentate chelator that would inactivate the iron redox cycle after its complexation, with a view to using this molecule in human therapy and/or in disease prevention. We synthesized such a chelator for the first time and show, by different physicochemical analysis, its tridentate nature and, importantly, its capacity to chelate iron with enough strength to inhibit both iron-dependent H(2)O(2) generation and lipid peroxidation in in vitro biological systems.     

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.     

Lead-induced iron overload and attenuated effects of ferroportin 1 overexpression in PC12 cells

Lead (Pb) neurotoxicity has received renewed interest with the growing evidence that Pb contributes to Alzheimer’s disease (AD). However, the mechanism is not clear. In our previous study of long-term Pb exposure in vivo, a brain iron (Fe) overload induced by Pb was observed in elderly rats. It is well known that brain Fe overload is the mechanism of AD. Therefore, we have reason to believe that Pb induced Fe overload and caused neurodegenerative disease. However, the mechanism or route of Pb-induced Fe overload is unknown. In the current study, the effect of Pb exposure on Fe homeostasis in PC12 cells was determined at different Pb-exposure concentrations and periods with differing Fe exposure, and the role of ferroportin 1 (FP1), the sole iron efflux protein, in Pb-induced Fe metabolic disorders was further investigated. The results showed a Pb-induced cellular increase in Fe accompanying a decrease in the expression of FP1 in a concentration- and time-dependent manner in Pb-exposed PC12 cells. Furthermore, FP1 overexpression could attenuate Fe accumulation in Pb-exposed PC12 cells. These results indicated that FP1 might be a novel target to prevent cellular Fe accumulation induced by Pb exposure and subsequent neurotoxic consequences.     

Cardiovascular aspect of Beta-thalassaemia

Beta-thalassaemia major is a genetic blood disorder caused by the reduced synthesis of beta globin chain. The consequences of the resulting chronic anaemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections, and hypercoagulability. Transfusional iron overload can affect heart function by directly damaging tissue through iron deposition or via iron-mediated effects at other sites. Cardiac dysfunction is common in patients with thalassaemia and is the leading cause of mortality. The main cardiac abnormalities reported in patients with thalassaemia major (TM) and iron overload are left ventricular systolic and diastolic dysfunction, pulmonary hypertension, valvulopathies, arrhythmias and pericarditis. These cardiac abnormalities are a consequence of the general co-morbid conditions in thalassaemia but are closely related to concomitant endocrine deficiencies, hypercoagulability state and inflammatory milieu. Iron's toxicity within cells arises from its capacity to catalyse the production of reactive oxygen species that cause lipid peroxidation and organelle damage, which lead ultimately to cell death and fibrosis. With the introduction of new technologies such as cardiac magnetic resonance T2* , the early detection of cardiac iron overload and associated cardiac dysfunction is now possible, allowing time for reversal through iron chelation therapy.     

Lead-induced iron overload and attenuated effects of ferroportin 1 overexpression in PC12 cells

Lead (Pb) neurotoxicity has received renewed interest with the growing evidence that Pb contributes to Alzheimer’s disease (AD). However, the mechanism is not clear. In our previous study of long-term Pb exposure in vivo, a brain iron (Fe) overload induced by Pb was observed in elderly rats. It is well known that brain Fe overload is the mechanism of AD. Therefore, we have reason to believe that Pb induced Fe overload and caused neurodegenerative disease. However, the mechanism or route of Pb-induced Fe overload is unknown. In the current study, the effect of Pb exposure on Fe homeostasis in PC12 cells was determined at different Pb-exposure concentrations and periods with differing Fe exposure, and the role of ferroportin 1 (FP1), the sole iron efflux protein, in Pb-induced Fe metabolic disorders was further investigated. The results showed a Pb-induced cellular increase in Fe accompanying a decrease in the expression of FP1 in a concentration- and time-dependent manner in Pb-exposed PC12 cells. Furthermore, FP1 overexpression could attenuate Fe accumulation in Pb-exposed PC12 cells. These results indicated that FP1 might be a novel target to prevent cellular Fe accumulation induced by Pb exposure and subsequent neurotoxic consequences.