Journal of Antimicrobial Chemotherapy

JAC Advance Access originally published online on August 23, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1070-1073; doi:10.1093/jac/dkl350

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Deferiprone iron chelation as a novel therapy for experimental mucormycosis

Ashraf S. Ibrahim^1,2,*, John E. Edwards, Jr^1,2, Yue Fu^1,2 and Brad Spellberg^1,2

¹ David Geffen School of Medicine at UCLA, Los Angeles CA, USA ² Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center Torrance, CA, USA

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^*Corresponding author. Tel: +1-310-222-6424; Fax: +1-310-782-2016; E-mail: ibrahim{at}labiomed.org

Received 16 May 2006; returned 14 July 2006; revised 3 August 2006; accepted 4 August 2006

	Abstract

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Objectives: Patients treated with the iron chelator deferoxamine are known to be more susceptible to mucormycosis. However, while deferoxamine is an iron chelator from the perspective of the human host, deferoxamine actually serves as a siderophore, delivering free iron to Rhizopus oryzae, the major cause of mucormycosis. Other iron chelators, including deferiprone, which do not deliver iron to R. oryzae have been described. We therefore sought to determine whether iron-chelation therapy with deferiprone would effectively treat mucormycosis.

Methods: In vitro MIC and minimum fungicidal concentration (MFC) of the iron chelator, deferiprone, for R. oryzae were determined by microdilution assay. In addition, we compared the efficacy of deferiprone with that of liposomal amphotericin B (LAmB) in treating mucormycosis in diabetic ketoacidotic mice.

Results: Deferiprone demonstrated static activity against R. oryzae at 24 h, but showed cidality at 48 h of incubation. Deferiprone was as effective as LAmB at improving survival and decreasing brain fungal burden, and both drugs were more effective than placebo in non-iron-overloaded animals. Administration of free iron with deferiprone reversed protection, confirming that the mechanism of protection was iron chelation.

Conclusions: Iron chelation is a promising, novel therapeutic strategy for refractory mucormycosis infections. Further studies are warranted to evaluate combination antifungal/iron chelation therapy and to evaluate the efficacy of other iron-chelating agents.

Keywords: zygomycosis , Rhizopus oryzae , mice

	Introduction

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Mucormycosis is a life-threatening infection caused by fungi of the class Zygomycetes, order Mucorales.¹ Rhizopus oryzae (Rhizopus arrhizus) is by far the most common cause of infection.¹ Typical conditions predisposing patients to developing mucormycosis include diabetic ketoacidosis, neutropenia, corticosteroid therapy, broad spectrum antibiotics, severe malnutrition and breakdown of cutaneous barriers.¹ Due to the increasing frequency of these risk factors, the incidence of mucormycosis has significantly increased over the past 2 decades.¹–⁴

Iron is required by virtually all microbial pathogens for growth and virulence.¹ For example, in mammalian hosts, sequestration of serum iron by carrier proteins such as transferrin is a major host defence mechanism against infection in general and R. oryzae in particular.¹,⁵ Rhizopus grows poorly in serum unless exogenous iron is added,⁵,⁶ and patients with elevated levels of available serum iron are uniquely susceptible to infection by R. oryzae and other Zygomycetes, but not to other pathogenic fungi.¹ For example, administration of deferoxamine or free iron worsens survival of animals infected with Rhizopus but not Candida albicans.⁶,⁷ While deferoxamine acts as an iron chelator with respect to the host, Rhizopus possesses specific receptors for deferoxamine that enable the organism to bind to iron-deferoxamine complexes, liberate the iron via an energy-mediated reductive process and then take up the iron.⁶,⁸ In vitro studies confirmed that Rhizopus accumulated 8- and 40-fold greater amounts of radiolabelled iron in the presence of deferoxamine than did Aspergillus fumigatus and C. albicans, respectively.⁶ This increased iron uptake by Rhizopus was linearly correlated with its growth in serum.⁶ Finally, patients treated with the iron chelator, deferoxamine, have a markedly increased incidence of invasive mucormycosis, which is associated with a mortality of >80%.⁹

Patients with diabetic ketoacidosis are also at high risk of developing rhinocerebral mucormycosis.¹ Importantly, these patients also have elevated levels of available serum iron, likely due to release of iron from binding proteins in the presence of acidosis.⁵

Because elevated available serum iron is integral for the virulence of mucormycosis, the use of an iron chelator that cannot be utilized by the fungus to scavenge iron from the host should prove to be efficacious against these infections. Deferiprone (1,2 dimethyl-3-hydroxy-4(1H)-pyridinone also known as L1, CP20, Ferriprox, or Kelfer) is a member of the -ketohydroxypyridine class of iron chelators and is approved for use in iron overload conditions in India and Europe.¹⁰,¹¹ In contrast to deferoxamine, deferiprone cannot be utilized by R. oryzae as a xenosiderophore.⁹ We therefore compared the efficacy of deferiprone with that of liposomal amphotericin B (LAmB) in treating mucormycosis in our diabetic ketoacidotic (DKA) mouse model. Here we demonstrate that deferiprone is an effective therapy for mucormycosis in the DKA mouse model. These findings suggest the need for further experimental and clinical studies evaluating the usefulness of iron-chelation therapy in combination with antifungals for the treatment of mucormycosis.

	Materials and methods

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R. oryzae and culture conditions

R. oryzae 99-880 was isolated from a brain abscess of a diabetic patient. The organism was grown on potato dextrose agar (PDA) for 3 days at 37°C. In some experiments, R. oryzae was starved for iron by growth on PDA in the presence of 1 mM ascorbic acid and ferrozine. The sporangiospores were collected in endotoxin-free PBS containing 0.01% Tween 80, washed with PBS and then counted with a haemocytometer to prepare the final inocula.

Susceptibility testing and animal model

MIC and minimum fungicidal concentration (MFC) were determined for deferiprone and deferoxamine by the method of Espinel-Ingroff using R. oryzae spores starved for iron.¹²

For in vivo infection, BALB/c male mice (20 g) were rendered diabetic with a single intraperitoneal injection of 210 mg/kg streptozotocin in 0.2 mL of citrate buffer 10 days prior to fungal challenge. Glycosuria and ketonuria were confirmed in all mice 7 days after streptozotocin treatment. Mice were infected through the tail vein with the appropriate inocula of R. oryzae. To confirm the inocula, dilutions were streaked on PDA plates and colonies were counted following a 24 h incubation period at room temperature. The primary efficacy endpoint was time to death. As a secondary endpoint, brain fungal burden (the primary target organ)¹³ was determined by homogenization by rolling a pipette on organs placed in Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) containing 2 mL of saline. The homogenate was serially diluted in 0.85% saline and then quantitatively cultured on PDA. Values were expressed as log₁₀ cfu/g of tissue. All procedures involving mice were approved by the institutional animal use and care committee, following the National Institutes of Health guidelines for animal housing and care.

Drugs and therapy regimens

LAmB diluted in 5% dextrose water was obtained from Gilead Sciences and was administered at 15 mg/kg/day intravenously through the tail vein. Deferiprone (Apotex Research Inc.) was dissolved in iron-free water and administered intraperitoneally once every day or every other day. Previous published and unpublished observations (John Connelly, Apotex Inc., Toronto, Canada, personal communication) have shown that repeated doses of 200 mg/kg of deferiprone, given once daily, results in significant toxicity (including some deaths) in non-iron-overloaded mice, guinea pigs and rats.¹⁴–¹⁶ Therefore, we chose 200 mg/kg/day to be the maximum dose and chose serial 2-fold decreases from there. In pilot studies, no efficacy was seen at doses below 50 mg/kg/day (data not shown). Therefore, 50, 100 and 200 mg/kg daily or every other day was studied. Treatment was begun 24 h post-infection and continued for a total of four doses. Control groups were treated with the diluent, 5% dextrose water.

In some experiments, a saturating dose of free iron was administered with deferiprone, in an attempt to reverse the efficacy of iron-chelation. Deferiprone is known to form molecular complexes with ferric iron (Fe³⁺) in a 1:3 ratio of iron to deferiprone.⁹ Based on the known molecular weights of ferric chloride (FeCl₃, molecular weight 162.22 g/mol) and deferiprone (molecular weight 139 g/mol) a 60 mg/kg dose of FeCl₃ was calculated to result in a significant excess of Fe³⁺ versus a 100 mg/kg dose of deferiprone given to an 18 g mouse.

Statistical analysis

The non-parametric log-rank test was used to determine differences in survival times of the mice. Differences in tissue fungal burdens in the infected organs were compared by the non-parametric Steel test for multiple comparisons. P values of <0.05 were considered significant.

	Results

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Deferiprone is highly active against R. oryzae in vitro

Initially, we determined the in vitro activity of deferiprone against R. oryzae. Deferiprone was static against R. oryzae at 24 h (MIC and MFC = 3.12 and 100 mg/L, respectively), but demonstrated cidality at 48 h of incubation (MIC and MFC = 6.25 mg/L). In contrast, deferoxamine, which is known to supply iron to R. oryzae,⁶ did not inhibit the growth of R. oryzae (MIC and MFC of >100 mg/L after 24 or 48 h), and in fact, by visual inspection, growth in wells containing deferoxamine was greater than in the growth control wells (containing no iron chelators).

Deferiprone protected DKA mice from R. oryzae infection

Having confirmed its in vitro activity against R. oryzae, we used our DKA mouse model to evaluate the role of deferiprone in treating disseminated R. oryzae infection in vivo.¹⁷ Initially, a dose response was performed, using doses based on unpublished observations from the manufacturer (50, 100 or 200 mg/kg of deferiprone administered once every day or every other day). Mice were infected with 4.3 x 10³ spores of R. oryzae and deferiprone treatment was initiated 24 h post-infection and continued for a total of four doses. A deferiprone dose of 100 mg/kg every other day improved survival of DKA mice compared with placebo (P = 0.027, Figure 1a). Higher doses, including 100 mg/kg administered every day or 200 mg/kg administered every day or every other day, did not improve survival, nor did the 50 mg/kg administered every day or every other day.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Deferiprone (Def) improves the survival of DKA mice with mucormycosis. Mice were treated 24 h post-infection with deferiprone, deferiprone plus ferric chloride (FeCl3, 60 mg/kg) to reverse the effect of iron chelation or LAmB, for a total of four doses. (a) Survival of DKA mice (n = 6 per group) infected with R. oryzae (4.3 x 103 spores) and treated with different treatment regimens of deferiprone. (b) Relation between deferiprone and iron and survival of R. oryzae-infected (5 x 103 spores) DKA mice (n 20 in each treatment). Experiments were performed at least twice on different days. *P < 0.05 compared with placebo. **P < 0.003 compared with placebo or deferiprone + FeCl3 by log-rank test.

 
Having established efficacy of deferiprone, we next compared its efficacy with a standard treatment of mucormycosis, high-dose LAmB. A dose of 15 mg/kg of LAmB administered every day was chosen because we demonstrated that this dose is more protective in our DKA mouse model than 1 mg/kg amphotericin B deoxycholate.¹⁷ Furthermore, to confirm that the mechanism of protection of deferiprone was chelation of iron, the efficacy of deferiprone plus a saturating dose (60 mg/kg) of free iron in the form of FeCl₃ was also evaluated. FeCl₃ was administered intraperitoneally every time deferiprone was given to the animals.

LAmB or deferiprone at 100 mg/kg administered every other day improved 28 day survival compared with placebo (Figure 1b, P < 0.003 by log-rank test for LAmB or deferiprone versus placebo). Survival rates for LAmB-treated mice were higher (52%) in comparison with deferiprone-treated mice (30%) at day 28; however, the difference was not significant (P = 0.15). The efficacy of deferiprone was completely abrogated by administration of ferric chloride (Figure 1b).

As an additional marker of efficacy, we evaluated the impact of deferiprone therapy on brain fungal burden, as the brain is the primary target organ in this model.¹³ Mice were infected with 3.8 x 10³ spores and then treated with two doses of LAmB (every day) or deferiprone (every other day). In the prior experiment, control mice began to die before the second dose of deferiprone had been administered. To enable testing of at least two doses of every-other-day deferiprone prior to determining tissue fungal burden, in this experiment deferiprone was administered at 30 min and 48 h post-infection, whereas LAmB was given 24 and 48 h post-infection. Brains were harvested 54 h post-infection. Both drugs reduced brain fungal burden compared with placebo (P 0.036) (Figure 2).

View larger version (6K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Brain R. oryzae burden of DKA mice (n = 6) treated with deferiprone, LAmB or placebo. Mice were infected with 3.8 x 103 spores and brains harvested 54 h later after two doses of treatment with either drug. Data are displayed as medians ± interquartile ranges. The y-axis reflects the lower limit of detection of the assay. *P 0.036 versus placebo by Steel test for multiple comparisons.

 

	Discussion

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In the absence of surgical removal of the infected focus (such as excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative for mucormycosis.¹ Furthermore, even when surgical debridement is combined with high-dose polyenes, the mortality associated with mucormycosis exceeds 50%.¹ In patients with disseminated disease mortality approaches 100%.¹ Because of this unacceptably high mortality rate, and the extreme morbidity of highly disfiguring surgical therapy, it is highly desirable to develop improved strategies to treat and prevent invasive mucormycosis.

Elevated available serum iron predisposes patients to mucormycosis.¹ Therefore it is logical to utilize suitable iron chelators to prevent or treat mucormycosis. Deferiprone is an iron chelator that cannot be used as a xenosiderophore by R. oryzae.⁹ Our susceptibility testing confirms that deferiprone potently inhibits the growth of R. oryzae in vitro, and it is actually cidal after 48 h of incubation. Furthermore, we demonstrated an impressive activity of deferiprone in protecting against highly lethal R. oryzae infection in the DKA mouse model. This activity was comparable to that of high-dose LAmB therapy, both with respect to survival and brain fungal burden.

Deferiprone also binds to aluminum, zinc and copper, but the stability of deferiprone complexes with these other cations is <50% of its binding stability with ferric iron, so the compound strongly favours interaction with iron over these other trace metals.¹⁸ Free-iron-mediated reversal of deferiprone protection confirmed that the mechanism of deferiprone protection against mucormycosis is iron chelation.

Our results are consistent with previously published data¹⁴–¹⁶ and indicate that deferiprone has a narrow therapeutic window in non-iron-overloaded animals. Specifically, neither doses above or below 100 mg/kg every other day improved survival. Doses above 100 mg/kg/day are known to be toxic, and we found doses lower than 100 mg/kg to be ineffective. Hence although our study provides proof-of-concept of iron-chelation therapy for mucormycosis, the narrow therapeutic window of deferiprone in the present study indicates that alternative dosing strategies/routes of administration, and alternative iron chelators, are worthy of investigation.

Although deferiprone is not licensed for use in North America, it has been administered to thousands of patients with iron overload in India and Europe¹¹,¹⁸ and is available in Canada and the United States on a compassionate use basis. Our results confirm that iron plays an important role in the pathogenesis of mucormycosis and that the use of iron chelators is a promising and novel therapeutic strategy for mucormycosis. Further investigation of the role of iron-chelation therapy in treating mucormycosis is warranted; specifically, alternative iron chelators and combination therapy including iron chelators and polyenes are currently under investigation.

	Transparency declarations

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None to declare.

	Acknowledgements

 
This work was presented in part at the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2005 (Abstract B-51). This work was supported by Public Health Service grants R01 AI63503-01A2 and R21 AI064716-01A2 and a research and educational grant to A. S. I. from Gilead Sciences Inc. B. S. is supported by Public Health Service grant K08 AI060641. J. E. E. is supported by R01 AI19990 and AI063382, and an unrestricted Freedom to Discover Grant for Infectious Disease from Bristol Myers Squibb. A. S. I. is also supported by a Burroughs Wellcome New Investigator Award in Molecular Pathogenic Mycology.

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This Article Abstract FREE Full Text (PDF) All Versions of this Article: 58/5/1070    most recent dkl350v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (22) Request Permissions Disclaimer Google Scholar Articles by Ibrahim, A. S. Articles by Spellberg, B. Search for Related Content PubMed PubMed Citation Articles by Ibrahim, A. S. Articles by Spellberg, B. Social Bookmarking          What's this?

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