Iron Metabolism in Aminolevulinic Acid-Photodynamic Therapy with Iron Chelators from the Thiosemicarbazone Group.

Iron plays a crucial role in various metabolic processes. However, the impact of 5-aminolevulinic acid (ALA) in combination with iron chelators on iron metabolism and the efficacy of ALA-photodynamic therapy (PDT) remain inadequately understood. This study aimed to examine the effect of thiosemicarbazone derivatives during ALA treatment on specific genes related to iron metabolism, with a particular emphasis on mitochondrial iron metabolism genes. In our study, we observed differences depending on the cell line studied. For the HCT116 and MCF-7 cell lines, in most cases, the decrease in the expression of selected targets correlated with the increase in protoporphyrin IX (PPIX) concentration and the observed photodynamic effect, aligning with existing literature data. The Hs683 cell line showed a different gene expression pattern, previously not described in the literature. In this study, we collected an extensive analysis of the gene variation occurring after the application of novel thiosemicarbazone derivatives and presented versatile and effective compounds with great potential for use in ALA-PDT.

ATH434, a promising iron-targeting compound for treating iron regulation disorders.

Cytotoxic accumulation of loosely bound mitochondrial Fe2+ is a hallmark of Friedreich's Ataxia (FA), a rare and fatal neuromuscular disorder with limited therapeutic options. There are no clinically approved medications targeting excess Fe2+ associated with FA or the neurological disorders Parkinson's disease and Multiple System Atrophy. Traditional iron-chelating drugs clinically approved for systemic iron overload that target ferritin-stored Fe3+ for urinary excretion demonstrated limited efficacy in FA and exacerbated ataxia. Poor treatment outcomes reflect inadequate binding to excess toxic Fe2+ or exceptionally high affinities (i.e. ≤10-31) for non-pathologic Fe3+ that disrupts intrinsic iron homeostasis. To understand previous treatment failures and identify beneficial factors for Fe2+-targeted therapeutics, we compared traditional Fe3+ chelators deferiprone (DFP) and deferasirox (DFX) with additional iron-binding compounds including ATH434, DMOG, and IOX3. ATH434 and DFX had moderate Fe2+ binding affinities (Kd's of 1-4  µM), similar to endogenous iron chaperones, while the remaining had weaker divalent metal interactions. These compounds had low/moderate affinities for Fe3+(0.46-9.59 µM) relative to DFX and DFP. While all compounds coordinated iron using molecular oxygen and/or nitrogen ligands, thermodynamic analyses suggest ATH434 completes Fe2+ coordination using H2O. ATH434 significantly stabilized bound Fe2+ from ligand-induced autooxidation, reducing reactive oxygen species (ROS) production, whereas DFP and DFX promoted production. The comparable affinity of ATH434 for Fe2+ and Fe3+ position it to sequester excess Fe2+ and facilitate drug-to-protein iron metal exchange, mimicking natural endogenous iron binding proteins, at a reduced risk of autooxidation-induced ROS generation or perturbation of cellular iron stores.

Application of validated UV-Vis spectrophotometry-colorimetric methods for specific quantification of deferiprone in the development of iron-responsive nanoparticle loaded into dissolving microneedle.

Deferiprone (DFP) is one of the iron-chelating agents used in iron overload therapy for patients with ß-thalassemia major (ß-TM). However, the use of DFP is limited as it experiences a first-pass effect and can potentially cause iron deficiency due to uncontrolled release. Therefore, iron-responsive (NP-IR) DFP nanoparticle innovation was developed to control DFP release. A dissolving microneedle system (NP-IR-DMNs) was used to maximize DFP release. However, in support of this development, validation of analytical methods using spectrophotometry and colorimetrics was carried out. UV-Vis spectrophotometry is an approach that is easy to use, practical, and more cost-effective than others. The DFP levels were determined in normal and iron-overloaded medium solutions with 1%, 2%, and 4% concentrations. In addition, DFP levels were also measured in rat plasma using the colorimetric method with the addition of FeCl3 reagent to increase sensitivity for the detection of the analyte. The procedures used as guidelines in the validation procedure are The International Council for Harmonization (ICH). As a result, all linear correlation values of medium and plasma ≥ 0.999 were obtained. The LOQ levels obtained were 0.55 µg/mL, 0.44 µg/mL, 0.42 µg/mL, 0.52 µg/mL, and 1.01 µg/mL in plasma, 1% FeSO4, 2% FeSO4, 4% FeSO4, and normal media, respectively. The accuracy and precision were confirmed valid, as all values were within the requirements and did not change during dilution. Then, this approach was successfully applied to determine the levels of DFP in NP-IR integrated into DMNs.

Amphetamine-like Deferiprone and Clioquinol Derivatives as Iron Chelating Agents.

The accumulation of iron in dopaminergic neurons can cause oxidative stress and dopaminergic neuron degeneration. Iron chelation therapy may reduce dopaminergic neurodegeneration, but chelators should be targeted towards dopaminergic cells. In this work, two series of compounds based on 8-hydroxyquinoline and deferiprone, iron chelators that have amphetamine-like structures, have been designed, synthesized and characterized. Each of these compounds chelated iron ions in aqueous solution. The hydroxyquinoline-based compounds exhibited stronger iron-binding constants than those of the deferiprone derivatives. The hydroxyquinoline-based compounds also exhibited greater free radical scavenging activities compared to the deferiprone derivatives. Molecular dynamics simulations showed that the hydroxyquinoline-based compounds generally bound well within human dopamine transporter cavities. Thus, these compounds are excellent candidates for future exploration as drugs against diseases that are affected by iron-induced dopaminergic neuron damage, such as Parkinson's disease.

Formation mechanism of blue pigment in boiled lotus rhizome discs: Insight into the chelation of polyphenols and iron.

Boiled lotus rhizome discs (BLRDs), as common processed products of lotus rhizome, have gained increasing attention from consumers and food manufacturers. However, the blue pigment formed during boiling affects its appearance and reduces the appetite of BLRDs. In this study, the effects of polyphenols and iron contents on blue pigment formation in BLRDs in different regions and months were investigated. Results revealed that blue variation was more serious in March and April of the second year in Wuhan, and polyphenols and iron contents in these two months were significantly higher than those in other months. Then, UPLC and UV-Vis analysis showed that polyphenols causing the formation of blue pigment in BLRDs were L-dopa, gallocatechin, catechin, epigallocatechin, chlorogenic acid and epicatechin, among which L-dopa (52.450 mg/100 g in fresh lotus rhizome (FLR)) and gallocatechin (36.210 mg/100 g in FLR) possessed the greatest effect. Moreover, the ESI-Q-TOF-MS analysis of L-dopa-iron chelate and gallocatechin-iron chelate suggested that the blue pigment of BLRDs was mainly in the form of bis-complexes under boiling conditions. The study on formation mechanism of blue pigment in BLRDs can provide a reference for lotus rhizome processing.

Chromone-deferiprone hybrids as novel MAO-B inhibitors and iron chelators for the treatment of Alzheimer's disease.

A series of chromone-deferiprone hybrids were designed, synthesized, and evaluated as inhibitors of human monoamine oxidase B (hMAO-B) with iron-chelating activity for the treatment of Alzheimer's disease (AD). The majority exhibited moderate inhibitory activity towards hMAO-B and potent iron-chelating properties. Particularly, compound 25c demonstrated remarkable selectivity against hMAO-B with an IC50 value of 1.58 µM and potent iron-chelating ability (pFe3+ = 18.79) comparable to that of deferiprone (pFe3+ = 17.90). Molecular modeling and kinetic studies showed that 25c functions as a non-competitive hMAO-B inhibitor. According to the predicted results, compound 25c can penetrate the blood-brain barrier (BBB). Additionally, it has been proved to display significant antioxidant activity and the ability to inhibit neuronal ferroptosis. More importantly, compound 25c reduced the cognitive impairment induced by scopolamine and showed significant non-toxicity in short-term toxicity assays. In summary, compound 25c was identified as a potential anti-AD agent with hMAO-B inhibitory, iron-chelating and anti-ferroptosis activities.

A novel antioxidant iron-chelating peptide from yak skin: analysis of the chelating mechanism and digestion stability in vitro.

BACKGROUND: The global prevalence of iron deficiency has posed significant public health risks. Animal-derived collagen peptides have been recognized for their potent metal ion-chelating capabilities, which can greatly enhance the bioavailability of iron. Yak skins, typically discarded during production and processing, serve as a valuable resource. Based on yak skin collagen peptide (YSP), we have developed a novel iron-chelating peptide: yak skin collagen iron-chelating peptide (YSP-Fe). RESULTS: The maximum level of iron chelation of YSP-Fe achieved was 42.72 ± 0.65 mg g-1. Structural analysis indicated that YSP-Fe was primarily formed from amino, carboxyl and carbonyl groups combined with ferrous ions. Through examination of the amino acid composition, molecular docking and peptide sequence identification, it was determined that Gly, Asp and Arg played crucial roles in the chelation of ferrous ions by YSP. Furthermore, YSP-Fe was more stable in simulated gastrointestinal digestion compared to FeSO4. CONCLUSION: YSP-Fe demonstrated dual benefits of iron supplementation and antioxidant effects. These significant findings provide a foundation for the development of novel iron supplements and the effective utilization of yak skin as a valuable resource. © 2024 Society of Chemical Industry.

Novel meroterpene-like compounds inhibit ferroptosis through Fe2+ chelation.

Colorectal cancer (CRC) is the third most common type of cancer in the world. It is characterized by complex crosstalk between various signaling pathways, as a result of which it is highly challenging to identify optimal therapeutic targets and design treatment strategies. In this study, we tested the effect of 700 compounds on the CRC cell line HT-29 by using the sulforhodamine B assay and screened out 17 compounds that exhibited high toxicity (indicated by an inhibition rate of ≥75 % when applied at a concentration of 10 µM) against the HT-29 cell line. Next, we investigated the mechanisms underlying the effects of these 17 highly toxic compounds. The results of ferroptosis analysis and electron microscopy showed that compounds 575 and 578 were able to significantly reverse RSL3-induced increase in ferroptosis, while compound 580 had a less pronounced ferroptosis-regulating effect. In subsequent experiments, western blotting showed that compounds 575, 578, and 580, which belong to a class of meroterpene-like compounds that affect ferroptosis, do not induce autophagy or apoptosis in the CRC cell line. Instead, Fe2+ chelation experiments showed that these three compounds can serve as iron chelators by chelating Fe2+ at a 1:1 (chelator: Fe2+) ratio. Specifically, the aldehyde and hydroxyl groups of the benzene ring in these compounds may chelate Fe2+, thus reducing Fe2+ levels in cells and inhibiting ferroptosis. These results indicate that these novel meroterpene-like compounds are potential therapeutic small-molecule candidates for targeting ferroptosis in tumors.

Improvement of in vivo iron bioavailability using mung bean peptide-ferrous chelate.

There is an increasing amount of research into the development of a third generation of iron supplementation using peptide-iron chelates. Peptides isolated from mung bean were chelated with ferrous iron (MBP-Fe) and tested as a supplement in mice suffering from iron-deficiency anemia (IDA). Mice were randomly divided into seven groups: a group fed the normal diet, the IDA model group, and IDA groups treated with inorganic iron (FeSO4), organic iron (ferrous bisglycinate, Gly-Fe), low-dose MBP-Fe(L-MBP-Fe), high-dose MBP-Fe(H-MBP-Fe), and MBP mixed with FeSO4 (MBP/Fe). The different iron supplements were fed for 28 days via intragastric administration. The results showed that MBP-Fe and MBP/Fe had ameliorative effects, restoring hemoglobin (HGB), red blood cell (RBC), hematocrit (HCT), and serum iron (SI) levels as well as total iron binding capacity (TIBC) and body weight gain of the IDA mice to normal levels. Compared to the inorganic (FeSO4) and organic (Gly-Fe) iron treatments, the spleen coefficient and damage to liver and spleen tissues were significantly lower in the H-MBP-Fe and MBP/Fe mixture groups, with reparative effects on jejunal tissue. Gene expression analysis of the iron transporters Dmt 1 (Divalent metal transporter 1), Fpn 1 (Ferroportin 1), and Dcytb (Duodenal cytochrome b) indicated that MBP promoted iron uptake. These findings suggest that mung bean peptide-ferrous chelate has potential as a peptide-based dietary supplement for treating iron deficiency.

Digestion and absorption characteristics of iron-chelating silver carp scale collagen peptide and insights into their chelation mechanism.

Iron deficiency is widespread throughout the world, supplementing sufficient iron or improving the bioavailability of iron is the fundamental strategy to solve the problem of iron scarcity. Herein, we explored a new form of iron supplement, iron chelates of silver carp scales (SCSCP-Fe) were prepared from collagen peptide of silver carp scales (SCSCP) and FeCl2·4H2O, the effects of external environment and simulated gastrointestinal digestive environment on the stability of SCSCP-Fe and the structural changes of peptide iron chelates during digestion were investigated. The results of in vitro iron absorption promotion showed that the iron bioavailability of SCSCP-Fe was higher than that of FeSO4. Two potential high iron chelating peptides DTSGGYDEY (DY) and LQGSNEIEIR (LR) were screened and synthesized from the SCSCP sequence by molecular dynamics and LC-MS/MS techniques. The FTIR results displayed that the binding sites of DY and LR for Fe2+ were the carboxyl group, the amino group, and the nitrogen atom on the amide group on the peptide. ITC results indicated that the chelation reactions of DY and LR with Fe2+ were mainly dominated by electrostatic interactions, forming chelates in stoichiometric ratios of 1:2 and 1:1, respectively. Both DY and LR had a certain ability to promote iron absorption. The transport of DY-Fe chelate may be a combination of the three pathways: PepT1 vector pathway, cell bypass, and endocytosis, while LR-Fe chelate was dominated by bivalent metal ion transporters. This study is expected to provide theoretical reference and technical support for the high-value utilization of silver carp scales and the development of novel iron supplements.

Rigid Fe(III) Chelate with Phosphonate Pendants: A Stable and Effective Extracellular MRI Contrast Agent.

A novel Fe(III) complex, Fe-tBPCDTA, was synthesized and explored as a potential contrast agent for MRI. Compared to established agents like Fe-EDTA and Fe-tCDTA, Fe-tBPCDTA exhibited moderate relaxivity (r1 = 1.17 s-1·mmol-1) due to its enhanced second-sphere mechanism. It also displayed improved kinetic inertness, lower cytotoxicity, and enhanced redox stability. In vivo studies demonstrated its function as an extracellular fluid agent, providing tumor contrast comparable to that of Gd-DTPA at a higher dosage. Complete renal clearance occurred within 24 h. These findings suggest Fe-tBPCDTA as a promising candidate for further development as a safe and effective extracellular MRI contrast agent.

Novel microbial fermentation for the preparation of iron-chelating scallop skirts peptides-its profile, identification, and possible binding mode.

Iron chelating peptides have been widely utilized as iron supplements due to their excellent absorption capacity, However, the high cost and cumbersome manufacturing process of these peptides significantly limit their industrial application. In this study, fermentation was used for the first time to prepare iron chelating peptides. Bacillus altitudinis 3*1-3 was selected as the most suitable strain from 50 strains. The hydrolysates of fermented scallop skirts showed excellent iron-chelating capacity (9.39 mg/g). Aspartic acid, glutamic acid, and histidine are crucial for the binding of peptides to ferrous ions. The heptapeptide (FEDPEFE) forms six binding bonds with ferrous irons. Compared with ferrous sulfate, peptide-ferrous chelate showed more stability in salt solution and simulated gastrointestinal juice (p < 0.05). Furthermore, the fermentation method could save >50% of the cost compared with the enzymatic method. The results can provide a theoretical basis for the preparation of ferrous-chelated peptides using the fermentation method.

Therapeutic Potential of Iron Chelators in Viral Diseases: A Systematic Review.

BACKGROUND: Iron chelators (ICs) have recently emerged as one of the new methods of treatment for viral infections. This study aimed to evaluate the efficiency and safety of natural ICs compared to synthetic ICs. Natural and synthetic ICs are the most common therapeutic agents tested for the treatment of viral infections. When evaluated against synthetic ICs, natural ICs are probably favored owing to their lower toxicity and safer properties. The main objective of the present systematic review was to assess the current evidence on the role of pharmacological mechanisms in the treatment of viral infections. METHODS: This study was designed as a systematic review in which search strategies were focused on two electronic databases, PubMed, and Scopus, between 2017 and 2021. A search filter with two subjects, "iron chelators" and "viral infection", was employed. RESULTS: According to the results, both natural and synthetic chelators had a considerable impact on the treatment of viral infections via various mechanisms, with natural ICs being the most extensively used. CONCLUSION: Natural and synthetic ICs exert their effects through different pharmacological mechanisms. Among these compounds, natural chelators are more widely used due to their safety, efficacy, and a wider range of applications.

Supramolecular Arrangement of Lignosulfonate-Based Iron Heteromolecular Complexes and Consequences of Their Interaction with Ca2+ at Alkaline pH and Fe Plant Root Uptake Mechanisms.

Previous studies have shown that natural heteromolecular complexes might be an alternative to synthetic chelates to correct iron (Fe) deficiency. To investigate the mechanism of action of these complexes, we have studied their interaction with Ca2+ at alkaline pH, Fe-binding stability, Fe-root uptake in cucumber, and chemical structure using molecular modeling. The results show that a heteromolecular Fe complex including citric acid and lignosulfonate as binding ligands (Ls-Cit) forms a supramolecular system in solution with iron citrate interacting with the hydrophobic inner core of the lignosulfonate system. These structural features are associated with high stability against Ca2+ at basic pH. Likewise, unlike Fe-EDDHA, root Fe uptake from Ls-Cit implies the activation of the main root responses under Fe deficiency at the transcriptional level but not at the post-transcriptional level. These results are consistent with the involvement of some plant responses to Fe deficiency in the plant assimilation of complexed Fe in Ls-Cit under field conditions.

Design of Tetrazolium Cations for the Release of Antiproliferative Formazan Chelators in Mammalian Cells.

Cancer cells generally present a higher demand for iron, which plays crucial roles in tumor progression and metastasis. This iron addiction provides opportunities to develop broad spectrum anticancer drugs that target iron metabolism. In this context, prochelation approaches are investigated to release metal-binding compounds under specific conditions, thereby limiting off-target toxicity. Here, we demonstrate a prochelation strategy inspired by the bioreduction of tetrazolium cations widely employed to assess the viability of mammalian cells. We designed a series of tetrazolium-based compounds for the intracellular release of metal-binding formazan ligands. The combination of reduction potentials appropriate for intracellular reduction and an N-pyridyl donor on the formazan scaffold led to two effective prochelators. The reduced formazans bind as tridentate ligands and stabilize low-spin Fe(II) centers in complexes of 2:1 ligand-to-metal stoichiometry. The tetrazolium salts are stable in blood serum for over 24 h, and antiproliferative activities at micromolar levels were recorded in a panel of cancer cell lines. Additional assays confirmed the intracellular activation of the prochelators and their ability to affect cell cycle progression, induce apoptotic death, and interfere with iron availability. Demonstrating the role of iron in their intracellular effects, the prochelators impacted the expression levels of key iron regulators (i.e., transferrin receptor 1 and ferritin), and iron supplementation mitigated their cytotoxicity. Overall, this work introduces the tetrazolium core as a platform to build prochelators that can be tuned for activation in the reducing environment of cancer cells and produce antiproliferative formazan chelators that interfere with cellular iron homeostasis.

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron(III ).

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing the iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UV-visible spectroscopy, mass spectrometry, and IR and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of the α-helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pep-cyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens.

The synthesis and properties of mitochondrial targeted iron chelators.

Iron levels in mitochondria are critically important for the normal functioning of the organelle. Abnormal levels of iron and the associated formation of toxic oxygen radicals have been linked to a wide range of diseases and consequently it is important to be able to both monitor and control levels of the mitochondrial labile iron pool. To this end a series of iron chelators which are targeted to mitochondria have been designed. This overview describes the synthesis of some of these molecules and their application in monitoring mitochondrial labile iron pools and in selectively removing excess iron from mitochondria.

Identification of high iron-chelating peptides with unusual antioxidant effect from sea cucumbers and the possible binding mode.

Iron deficiency anemia (IDA) is a common nutritional disease affecting 2 billion people. To develop a new iron-fortified food, we designed a novel type of iron-chelating peptide [Sea cucumbers peptides (SCP)-Fe] from sea cucumbers. SCP can chelate ferrous ions. The neutral protease hydrolysate have the highest iron chelating activity (117.17 ± 2.62 mg/g). Single factors including pH, material ratio, and molecular weight, had a significant effect on the iron chelating activity. The characterization of the SCP-Fe chelate revealed a loose and blocky structure with increased particle size. The amino acid composition, peptide identification and molecular docking indicated that Asp, Glu, Gly and Pro played an important role in binding to ferrous ions. After chelation, SCP-Fe chelate had dual nutrition effects of stronger radical scavenging ability and potential high-efficiency iron supplementation ability. These results might provide insights into the methods for developing functional foods such as iron-fortified seafood.

Thiol-Reactive Arylsulfonate Masks for Phenolate Donors in Antiproliferative Iron Prochelators.

Tridentate thiosemicarbazones, among several families of iron chelators, have shown promising results in anticancer drug discovery because they target the increased need for iron that characterizes malignant cells. Prochelation strategies, in which the chelator is released under specific conditions, have the potential to avoid off-target metal binding (for instance, in the bloodstream) and minimize unwanted side effects. We report a prochelation approach that employs arylsulfonate esters to mask the phenolate donor of salicylaldehyde-based chelators. The new prochelators liberate a tridentate thiosemicarbazone intracellularly upon reaction with abundant nucleophile glutathione (GSH). A 5-bromo-substituted salicylaldehyde thiosemicarbazone (STC4) was selected for the chelator unit because of its antiproliferative activity at low micromolar levels in a panel of six cancer cell lines. The arylsulfonate prochelators were assessed in vitro with respect to their stability, ability to abolish metal binding, and reactivity in the presence of GSH. Cell-based assays indicated that the arylsulfonate-masked prochelators present higher antiproliferative activities relative to the parent compound after 24 h. The activation and release of the chelator intracellularly were corroborated by assays of cytosolic iron binding and iron supplementation effects as well as cell cycle analysis. This study introduces the 1,3,4-thiadiazole sulfonate moiety to mask the phenolate donor of an iron chelator and impart good solubility and stability to prochelator constructs. The reactivity of these systems can be tuned to release the chelator at high glutathione levels, as encountered in several cancer phenotypes.

Safety and efficacy of a novel iron chelator (HBED; (N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid)) in equine (Equus caballus) as a model for black rhinoceros (Diceros bicornis).

While iron overload disorder (IOD) and related disease states are not considered a common occurrence in domestic equids, these issues appear prevalent in black rhinoceroses under human care. In addressing IOD in black rhinos, altering dietary iron absorption and excretion may be the most globally practical approach. A main option for treatment used across other species such as humans, is chelation therapy using iron-specific synthetic compounds. As horses may serve as an appropriate digestive model for the endangered rhinoceros, we evaluated the potential use of the oral iron chelator N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) in horses for safety and efficacy prior to testing in black rhinoceros. Health and iron digestibility and dynamics were assessed in horses (n = 6) before, and after treatment with HBED (50 mg/kg body weight) for 8 days using a crossover design with serum, faecal and urine collection. A preliminary pharmacokinetic trial was also performed but no trace of HBED was found in serially sampled plasma through 8 h post-oral dosing. HBED increased urinary iron output in horses compared to control by 0.7% of total iron intake (p < 0.01), for an average of 27 mg urinary iron/day, similar to human chelation goals. Blood chemistry, blood cell counts and overall wellness were not affected by treatment. As healthy horses are able to regulate iron absorption, the lack of change in iron balance is unsurprising. Short-term HBED administration appeared to be safely tolerated by horses, therefore it was anticipated it would also be safe to administer to black rhinos for the management of iron overload.