Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

Metal overload plays an important role in several diseases or intoxications, like in Wilson's disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.     

Tetrathiomolybdate causes formation of hepatic copper-molybdenum clusters in an animal model of Wilson's disease

Wilson's disease is an autosomal recessive human illness in which large quantities of copper accumulate in various organs, including the brain and the liver. If left untreated, it results in hepatitis, neurological complications, and death. Long-Evans Cinnamon (LEC) rats have a homologous mutation to Wilson's disease and thus provide an animal model. Liver lysosomes from tetrathiomolybdate-treated LEC rats were isolated and analyzed by Cu and Mo K-edge X-ray absorption spectroscopy. The lysosomes contained a Cu-Mo-S cluster in which the Mo is coordinated by four sulfurs at 2.24 A with approximately three copper neighbors at 2.70 A. Each Cu is coordinated to 3-4 sulfurs at 2.28 A with approximately one Mo neighbor at 2.70 A. These results indicate the formation of a biologically novel molybdenum-copper-sulfur cluster.     

Synthesis and characterization of 8-aminoquinolines,substituted by electron donating groups,as high-affinity copper chelators for the treatment of Alzheimer's disease

The deregulation of copper homeostasis generates copper–amyloid aggregation and strongly participates in neuron damage in the brains of patients with Alzheimer's disease. Therefore, copper chelators able to regulate copper homeostasis should be considered as potential therapeutic agents. On the basis of a bidentate amine side chain attached at the 2-position of an 8-aminoquinoline motif, a series of low-molecular-weight copper chelators have been designed to act as specific tetradentate Cu(II) chelators. The affinity of these ligands for Cu(II) and their selectivity for Cu(II) with respect to Zn(II) are reported. These ligands provide a square planar tetradentate coordination sphere that should be suitable to extract copper (II) from copper–amyloid complexes, and are therefore expected to regulate copper homeostasis in vivo.     

肝豆状核变性病与人体内铜的关系

为探讨肝豆状核变性发病机理与人体内铜的关系，结合本实验室分析条件，用原子吸收分光光度法对509名患者的24小时尿铜、血清铜含量进行了检测。结果表明，109名患者的24小时尿铜含量远高于正常人水平，血清铜含量低于正常人水平，经诊断证实这些患者患有肝豆状核变性病。本文进一步对这些患者按不同年龄、不同性别进行了分析，为肝豆状核变性病的成因、发展、诊断及治疗提供参考依据。     

Simple and sensitive determination of hepatic copper by use of an extractive catalytic method

For determination of copper in liver tissue and in liver biopsy samples an extractive catalytic determination is proposed. After digestion of the liver samples with nitric acid, copper is extracted as the salicylate-pyridine complex into chloroform and determined catalytically directly in the organic extract by addition of ethanolic solutions of sulphanilic acid, pyridine and hydrogen peroxide. Copper can be determined in the range from 10 to 350 ppm (dry weight basis) with a relative standard deviation of 5-15%. The method enables copper to be determined in liver biopsy samples of about 5 mg dry weight by use of a simple spectrophotometer, and can be used in diagnosis of Wilson's disease.     

Coordination chemistry and biology of chelators for the treatment of iron overload disorders

Treatment of the medical condition generally referred to as iron overload through the delivery of chelators has recently received a major boost. In 2005 Novartis gained FDA approval for the drug deferasirox, which may be taken orally. Until this time most patients with Fe overload have had to endure long periods of subcutaneous infusions of the orally ineffective drug desferrioxamine (desferal) which has led to major problems with patient compliance. An effective Fe chelator must possess a number of properties for it to be able to complex Fe in vivo and be excreted intact. This Perspective will provide an overview of the current state of chelators for Fe overload; both those currently approved and those undergoing preclinical development.     

Challenges in chelating positron emitting copper isotopes: tailored synthesis of unsymmetric chelators to form ultra stable complexes

The synthesis of chelators that form high stability complexes with copper(II) isotopes and do not suffer from transchelation in vivo has been a goal for many chemists. Such chelators will facilitate the exploitation of the (64)Cu isotope (t(1/2) = 12.7 h, β(+) (19%); β(-) (39%); EC (41%)) for positron emission tomography imaging studies, which has a longer half life relative to the more commonly used (18)F (t(1/2) = 109.8 min) and (11)C (t(1/2) = 20.4 min) isotopes. One option is the CBTE2A chelator, which has been championed by Weisman, Wong and Anderson, and, more recently, alternate bifunctional chelator (BFC) versions have been synthesised. Improved synthetic methods are required for unsymmetric derivatisation of these chelators to allow more selective biomolecule attachment. This work investigates synthetic routes to form new unsymmetric chelating ligands via stepwise reaction of the bisaminal precursor, determines their X-ray structures and demonstrates cold copper(II) isotope complex formation.     

Iron chelating agents for the treatment of iron overload

The importance of iron chelators in medicine has significantly increased in recent years. Iron is essential for life but it is also potentially more toxic than other trace elements. This is because we lack effective means to protect human cells against iron overload and because of the role of iron in the generation of free radicals. In order to protect patients from the consequences of iron toxicity, iron chelating agents have been introduced in clinical practice. Unfortunately, the ideal chelator for treating iron overload in humans has not been identified yet. In this paper we examine a few characteristics of iron chelators, with some emphasis on the effects of redox cycling, on absorption mechanisms and on some properties of the pFe. A brief summary is then made of the chelators recently proposed or in development for the treatment of iron overload.     

Zinc in gastrointestinal and liver disease

Zinc is an essential trace element with important biological functions, depending on the structural and/or catalytic role played by zinc ions in a large variety of enzymes. Zinc plays a critical role in cellular integrity, protein synthesis, nucleic acid metabolism, contributing to cell growth, proliferation, differentiation and death. The present review reports data on zinc homeostasis and metabolism, zinc absorption, intercellular trafficking, intracellular transport inside enterocytes and hepatocytes. Particular emphasis is given to data regarding the role of zinc carriers ZnTs and Zips, and to their expression in liver and gut in experimental and in human studies. The role of zinc in the gastrointestinal tract and in the liver as a powerful antioxidant and its relationship with apoptosis is discussed. Possible implications of zinc status in different disorders of the gastrointestinal tract are presented, focusing on its possible introduction in the therapy of inflammatory bowel diseases. Data on the role of zinc and zinc carriers in the evolution of liver fibrosis towards cirrhosis are also discussed. Finally, data on the ability of zinc therapy to obtain regression of liver cirrhosis in patients affected by Wilson's disease are reported, and the hypothesis that zinc could protect against liver fibrosis in chronic liver disease of different origin is presented.     

Nonabsorbable Iron(III) binding polymers: Synthesis and evaluation of the chelating properties

Iron is a key micronutrient essential for many biological events. While iron deficiency can lead to anemia, supplementation with oral iron often ends up with enteral iron overload, a critical gastrointestinal (GI) burden linked to the increased risk of dysbiosis, infections and often associated with colorectal cancer. Iron chelation therapy is clinically used to reduce pathological systemic iron overload by established low molecular weight iron chelators. As drawbacks, these drugs present low pharmacokinetic profiles and several toxicities, leading to relatively high rates of adverse effects. To overcome these issues, the prevention of iron accumulation in the GI tract by non-absorbable iron binding polymers could represent an alternative still underexploited approach. Here, we present the development of a series of insoluble polymeric Fe(III) chelators. These innovative compounds have been obtained by the conjugation of 3-hydroxypyridin-4-one Fe(III) chelating moiety with branched Polyethyleneimine (PEI) and Carboxymethyl cellulose (CMC). In vitro binding studies indicated that the Fe(III) chelating capacity depends on the nature of the polymer. In particular, PEI derivatives possess higher selectivity toward Fe(III) in simulated intestinal fluid preserving the integrity of intestinal enterocytes, representing thus promising compounds in the development of iron chelators.     

Design of iron chelators with therapeutic application

Iron is essential for the proper functioning of all living cells, however it is toxic when present in excess. Thus, using iron chelators as therapeutic agents, namely chelation therapy, has received increasing attention. The objective of this review is to discuss the factors which should be considered when designing clinically useful iron chelators, to present the application of iron chelators in the treatment of iron overload associated with β-thalassaemia major and sickle cell anaemia, and to highlight the potential applications in the treatment of neurodegenerative disorders and microbial infection. This article reviews recent knowledge centred on these themes and indicates the growing importance of the concept of iron chelation in medicine.     

Protective effects of baicalin and quercetin on an iron-overloaded mouse: comparison of liver, kidney and heart tissues

Excessive accumulation of iron in the body can lead to organ injury. Some flavonoids which possess high affinity to iron can be used as iron chelators for curing iron overload diseases such as haemochromatosis. In this article, the effects of baicalin and quercetin on different mouse tissues (liver, kidney and heart) afflicted with iron overload-induced injury were studied. It was found that after administration of 500 mg kg?1 iron dextran for 45 days, the degree of oxidative injury in the three organs was different. When iron dextran-induced iron-overloaded mice were fed a diet containing baicalin or quercetin (1% w/w), both flavonoids showed significant effects in suppressing iron overload-induced injury. The efficiencies of both flavonoids on different tissues varied. Both flavonoids caused a significant decrease of iron content in liver and kidney tissues but not in the heart, whereas the flavonoids were more efficient in inhibiting the increase of carbonyl content and the thiobarbituric acid reactive substance levels in heart tissues. The results indicate that the protective effects of baicalin and quercetin on different tissues of iron-overloaded mice are different.     

Development of lactobionic acid conjugated-copper chelators as anticancer candidates for hepatocellular carcinoma

Hepatic copper deposition leads to metabolic disorders, rapid increase in reactive oxygen species (ROS) levels, and even the occurrence and metastasis of hepatocellular carcinoma. Copper chelation or copper transporter inhibition have already been developed into an effective method to control the canceration of hepatocytes and kill the hepatocarcinoma cells. Here, we designed three novel lactobionic acid conjugated copper chelators (GT1, 9 and 10), which have the potential to be recognized by asialoglycoprotein receptor (ASGPR), a high-capacity C-type lectin receptor selectively expressed in liver. Both GT1, 9 and 10 can selectively and efficiently coordinate with copper in solution and in the high-copper treated hepatocellular carcinomas model (HC HepG2 cells). The thiosemicarbazone-based chelator GT1 should more effectively eliminate copper and promote apoptosis of HC HepG2 cells, which might have application prospects in preventing cancerization and other pathological lesions caused by copper deposition of liver. Moreover, our results also revealed the potential of GT1 to be harnessed as preventive leading structures of the hepatocellular carcinomas.     

In Vivo Detection of Copper Ions by Magnetic Resonance Imaging Using a Prion-Based Contrast Agent

Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson??s disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO₄ solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.     

A theoretical study on the electronic structures and equilibrium constants evaluation of Deferasirox iron complexes

Elemental iron is essential for cellular growth and homeostasis but it is potentially toxic to the cells and tissues. Excess iron can contribute in tumor initiation and tumor growth. Obviously, in iron overload issues using an iron chelator in order to reduce iron concentration seems to be vital. This study presents the density functional theory calculations of the electronic structure and equilibrium constant for iron-deferasirox (Fe-DFX) complexes in the gas phase, water and DMSO. A comprehensive study was performed to investigate the Deferasirox-iron complexes in chelation therapy. Calculation was performed in CAMB3LYP/6-31G(d,p) to get the optimized structures for iron complexes in high and low spin states. Natural bond orbital and quantum theory of atoms in molecules analyses was carried out with B3LYP/6-311G(d,p) to understand the nature of complex bond character and electronic transition in complexes. Electrostatic potential effects on the complexes were evaluated using the CHelpG calculations. The results indicated that higher affinity for Fe(III) is not strictly a function of bond length but also the degree of Fe–X (X = O,N) covalent bonding. Based on the quantum reactivity parameters which have been investigated here, it is possible reasonable design of the new chelators to improve the chelator abilities.     

Copper-related diseases: From chemistry to molecular pathology

The aim of this review is to give a general view on the current status of the scientific basis for the role of copper in human health and disease, outlining the roles of copper in human metabolism and bioenergetics, its coordination chemistry as well as its biological ligands involved in the multiple steps of metal assimilation and distribution. Copper bioavailability depends on four main factors: (1) the absorption of copper from the gastrointestinal tract; (2) copper transport in the portal blood; (3) the extraction of copper by hepatocytes from the portal blood supply; (4) copper uptake by peripheral tissues and by the central nervous system. The most important copper pumps, providing a permeation pathway for copper ions in man, such as hCTR1, hCTR2, DMT1, ATP7A, ATP7B, and MURR1, are extensively discussed. Different theories on the putative role of ceruloplasmin in copper transport in blood are presented. Data on interactions among other trace elements and copper, from the level of enterocyte to other systems in the body, are also shown. Finally, the function of different drugs, chelators and non-chelating agents, utilized in clinical practice in the therapy of Wilson's disease is treated, in particular the “reductive chelation” of penicillamine is discussed.     

Chelating agents for human diseases related to aluminium overload

The aim of this review is to give a general view on the current status of the role of aluminium in human health and disease. The main aspects of aluminium metabolism in humans are covered, summarizing the state of knowledge on the absorption, distribution, retention and excretion of aluminium, giving particular emphasis to the main metabolic pathways of this metal ion in different organs. Then the pathological consequences of aluminium overload in man, its role in neurodegenerative diseases and the emerging implication of this metal ions in different pathologies are treated. Finally, the function of different chelators utilized in clinical practice in the therapy of aluminium depending diseases is discussed and the latest studies on aluminium chelators are presented. Some emphasis is given to the parallelism between iron and aluminium chelators, and in particular interesting correlations between structural parameters of these two trivalent metal ions are presented.     

Development of new amphiphilic bio-organic assemblies for potential applications in iron-binding and targeting tumor cells

Iron overload has been implicated in the pathogenesis of many neurodegenerative diseases, cancer and thalassemia. In this work, we have developed new supramolecular assemblies as potential iron chelators for mitigating iron overload at the cellular level. We utilized fluorenyl-based building blocks that were functionalized with ethylene diamine (Fmoc-Ed) or arginine (Fmoc-Arg). Fmoc-Ed was further conjugated with ureido propionic acid (UDP) or pyrazole-3-carboxylic acid (PCA). Each of the building blocks were self-assembled into nanovesicles or fibers and further functionalized with the transferrin receptor binding peptide THRPPMWSPVWP (Tf) to promote receptor mediated cellular uptake. Our results indicated that the assemblies were able to target HeLa cells, induce apoptosis, ROS formation and were able to penetrate the cells. The degree of cellular impact was dependent upon the structure of the assemblies. The effects were more prominent under iron overload conditions compared to normal growth conditions. Our results suggest that such nanoscale assemblies may open new avenues for further studies into iron chelation and mitigation of iron overload using Fmoc-functionalized building blocks.     

糖尿病人血清与尿中微量元素变化与营养保健的探讨

通过对１１７例糖尿病人血清及尿液分析：发现血清中铜、铁、锌、镁四元素明显低于正常人对照组，差异极为显著（Ｐ＜０．００１）；尿中２４小时排出总量除铁元素无差异外，其他三元素都明显高于正常人对照组（Ｐ＜０．００１）。铜、锌的排出总量是正常人２倍以上，伴随糖尿病增加重随之进一步增加，而血清中铜、锌的含量却随之进一步减少，呈负相关趋势．进而又加重了体内微量元素的缺乏．铜、锌是人体多种酶数的辅基成份或激活因子，对丘脑至靶器官等各级内分泌组组分泌激素有着重要的作用．在常规治疗中补充铜、锌营养对促进糖尿病患者的康复有重要的作用．