Abstract
Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3”-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high µM range. Here we show that FBS0701 has inhibition concentration 50% (IC50) of 6 µM for Plasmodium falciparum in contrast to the IC50 for deferiprone and deferoxamine at 15 and 30 µM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials.
Introduction
Iron metabolism is a proven target for many malaria drugs. The quinolines like chloroquine and quinine interfere with iron protoporphyrin IX crystallization in the digestive vacuole [1]. The artemisinins are activated by iron to generate carbon-centered radicals that rapidly kill parasites [2]. Iron chelators have been explored as alternative malaria drugs for decades [3]. Intravenous deferoxamine does increase clearance of parasites [4] but in an important clinical trial deferoxime did not effect outcome from severe cerebral malaria [5]. The orally available deferiprone was also suboptimum in an uncomplicated malaria trial because of poor iron clearance after oral absorption [6]. Iron regulation is potent in limiting liver stage multiplication mediated, in part, by hepcidin pathway [7]. Iron supplementation increases hepatic parasites [8] and iron chelators decrease hepatic parasites [7]. For intraerythrocyte Plasmodium, the literature describes chelation of both labile iron pools in parasites as well as infected erythrocyte cytosol. Deferoxamine decreases available iron in the nucleus and parasite cytosol. The exact mechanism of parasite killing is not known but evidence points to a iron chelator mechanism of limiting iron for ribonucleotide reductase production of nucleotides, however earlier studies also suggest a toxic mechanism of erythrocytic Plasmodium killing [3]. A drawback for Plasmodium iron chelation chemotherapy is interference with the parasiticidal action of artemisinin drugs [9], [10].
The potent novel chemical analogue of the bacterial siderophore desferrithiocin is entering clinical trials for transfusional iron overload. (S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]4-methyl-4-thiazolecarboxylic acid also known as (S)3”-(HO)-desazadesferrithiocin-polyether [DADFT-PE] or more simply as FBS0701 has a very high affinity and specificity for Fe (III) with an equilibrium constant of 10−22 Molar [11]. The molecular weight of the formulated salt is 441 while free base is 400. The iron clearing efficiency in rodents is 10.6+4.4% and in primates is 23+4.1% [11], [12]. In contrast the iron clearing efficiency for deferoxamine is 2.8% and 5.5% and deferiprone is 1.2% and 2.1% in rodents and monkeys, respectively. In normal human volunteers dosed with FBS0701 with 6, 10 or 16 mg/kg, the pharmacokinetics across the dosing range indicates high bioavailability Cmax of 22.7, 41.5 and 87.0 µM respectively, dose-proportionality and a terminal t1/2 permitting once-daily dosing [13].
Here we investigated the actions of this iron chelator on P. falciparum blood stage parasites, its action against gametocytes and efficacy in murine malaria models.
Methods
Materials
FBS0701 was obtained from Aptuit (Kansas City) LLC under good manufacturing practice. All other reagents and chemicals are from Sigma unless otherwise noted. P. falciparum isolates 3D7, Indo and KMVII were obtained from ATCC-BEI MR4 repository.
P. falciparum culture
Direct effect of FBS7010 on P. falciparum was determined by SYBR green I (Invitrogen-Molecular Probes, Eugene OR) based assay as described earlier [14]. Briefly, the inhibition assay was initiated by adding serially diluted FBS7010 in 96 well microplates followed by sorbitol synchronized 1% ring stage parasites at 1% hematocrit in RPMI 1640 media with glutamine and HEPES supplemented with 10% human serum and 50 mg/ml hypoxanthine. Culture plates were then transferred to 37°C incubator and maintained in the environment of 5% O2, 5% CO2 and 90% N2 for 72 hrs. 2× SYBR Green I solution in lysis buffer was added after one cycle of freeze and thaw. Plates were then placed at room temperature in dark for 1–2 hours and fluorescence values were quantitated in a plate reader (HTS 7000, Perkin Elmer) at emission and excitation wavelengths of 535 and 485, respectively. For fractional inhibition analysis quinine, quinidine, chloroquine and artemisinin were used at concentrations ranging from 1 to 100 nM and FBS0701 from 1–15 uM. The fractional inhibition was analyzed according to the original Elion and Hitchings paper [15] with modifications by Bell [16]. The fraction of the chloroquine, quinine or artemisinin drug in combination with FBS0701 that produced an IC50 was divided by the IC50 of the chloroquine, quinine or artemisinin alone for the ratio on the y-axis. The x-axis was fraction of FBS0701 in combination with the chloroquine, quinine or artemisinin divided by concentration of FBS0701 which produced an IC50.
To test the effect of FBS0701 on gametocyte stages, P. falciparum NF54 cultures were initiated in 24 well plates at 0.5% asexual parasitemia and 4% hematocrit [17]. Medium was changed daily up to day 18, without addition of fresh RBCs. Continuous cultivation without dilution leads to concomitant crash of asexual parasitemia and induction of gamteocytogenesis by day 5. The gametocytemia was approximately 7% at start of treatment. FBS0701 was dosed in wells at Days 9–10 , when majority of gametocytes were in early stage of development (Stage I, II) or Days 14–15 when majority of gametocytes have matured to stage III to V. Levels of gametocytemia were determined on day 18 and the mean number of gametocytes was calculated by counting 10 high-powered (1000×) fields from triplicate wells per condition. More than 1000 erythrocytes were enumerated by random scanning across Giemsa blood film.
Animal Ethics
All animal experiments were performed on protocol “Malaria Drug Testing in Mice” (Approval ID-MO09H401) approved by The Johns Hopkins Animal Care and Use Committee in accordance with institutional standards.
Malaria Murine Drug Testing
Male 5–6 week old at approximately 25±2 g C57/BL6 mice were purchased from Jackson Laboratories (Maine, USA). The P. berghei ANKA strain was obtained from ATCC; the P. yoelii 17× lethal strain was the gift of James Burns (Drexel University). Freshly made solutions of FBS0701 in water were used for all the experiments and drug was administered by oral cannulation at indicated times. Murine infection was initiated by intraperitoneal inoculation of a million parasites for P. berghei and 10 million for P. yoelii. Blood was taken from the tail vein for blood smears. Parasitemias were determined in a blinded fashion by counting four fields of approximately 200 erythrocytes per field. Mice that survived for 30 days post infection with complete disappearance of parasitemia and no recrudescence within the next 30 days were considered cured.
Pharmacokinetics
30 Balb/c mice were dosed with 100 mg/kg of FBS0701 by oral cannulation and blood was drawn by cardiac puncture during anesthesia. Whole blood was separated into plasma and erythrocytes and plasma sent for analysis by Covance Laboratories (Madison, WI) using a validated assay [13].
Measurement of Bioavailable Labile Iron in Erythrocytes
Blood was collected from healthy subjects washed 2× with PBS. Erythrocytes were then incubated with 0.125 uM calcein (Invitrogen) in PBS for 15 minutes at 37C, washed 2× with PBS and then allowed to rest for 10 minutes at 37C in the dark [18], [19], [20]. Cells were incubated with PBS, 100 µM FBS0701 or 100 µM diferiprone for 1 hour at 37C. Cells were analyzed by flow cytometry using a modified FACS-Calibur with 2 lasers 30 mW 488 Diode Pumped Solid State laser and a 25 mW 637 red diode laser (FACS-Calibur; Becton Dickinson, Mount View CA, modified by Cytek Development). Data was collected using FlowJo CE and analyzed using Summit v4.3.01. The emission of 400,000 cells was analyzed using logarithmic amplification for fluorescence signal height (FL-1, 530/30) and linear amplification for forward scatter (FSC) and side scatter (SSC). The threshold was set at FSC to exclude cell debris and microparticles. The mean fluorescence intensity (MFI) was calculated using Summit v4.3.01. Individual experiments were performed in triplicate and also biologic duplicate with different human erythrocyte donors. The statistical significance was calculated using the Student’s t-test.
Results
P. falciparum Blood Stage Inhibition
In the chloroquine and quinine sensitive isolate 3D7, the IC50 with continuous drug exposure was 6 µM (IC5 = 3 µM and IC90 = 10 µM). Testing in the chloroquine and quinine resistant parasites Indo and KMVII showed a similar inhibition to 3D7. Morphologically the FBS0701 exposed parasites were shrunken. Stage and concentration dependence of FBS0701 iron chelation showed that for continuous drug exposure starting at ring and trophozoite stage the IC50 was similar at 6 µM, but when drug exposure was initiated at schizont stage after DNA replication, the IC50 increased by almost three fold to 15–17 µM. Investigation of dose-dependent killing of gametocytes shows that 100 µM inhibited more than 90% of early stage I and II gametocytes but only about 40% of late stage III or IV gametocytes, 10 µM FBS0701 had minimal 37% decrease in the number of gametocytes (Figure 1). Because of the known interactions of iron chelators with the artemisinins [9], [16], [21], we investigated interaction in vitro with P. falciparum. Fractional inhibition indicates interference with artemisinin and an additive action with the quinolines like chloroquine or quinine (Figure 2).
Murine Malaria Inhibition
In the lethal P. bergheiANKA mouse model, FBS0701 showed a 50% reduction in day 5 parasitemia and delayed death by more than ten days at the 100 mg/kg dose, which was independent of single day dosing for one day, three days or seven days (not shown). The lethal P. yoelii model was used next because while lethal infection results from invasion of both normocytes and reticulocytes a recent vaccine study demonstrated that by largely restricting invasion to reticulocytes the infection was no longer lethal. [22]. We dosed mice with the FBS0701 by single oral dose either seven days or one day prior to parasite inoculation and also dosed a day after inoculation. All infected untreated control mice died by day 11. All the mice treated a day after infection with a single oral dose of the iron chelator survived (Figure 3). Surprisingly two to three of mice with FBS0701 pretreatment one or seven days prior to intraperitoneal P. yoelii infection still lived suggesting a lingering effect after the drug was cleared. In this model we again saw a small reduction in day 3 parasitemia. In all surviving mice, which had been dosed with FBS0701, the day 16 parasitemia was near 50% but confined to reticulocytes rather than normocytes (Figure 4). Resolution of parasitemia coincided with return of reticulocyte count to normal levels.
Mouse Pharmacokinetics
We performed a pharmacokinetic analysis of FBS0701 dosing in fed mice without malaria. 100 mg/kg dosing resulted in 17 µM levels at one hour that by 8 hours was close to 1 µM (Figure 5). These concentrations are below those easily achieved in humans [13].
Removal of Labile Erythrocyte Iron
Calcein has been used as an intracellular iron probe [23], [24]. Labile iron bound to low molecular weight, low affinity chelators binds and quenches calcein to generate the baseline equilibrium fluorescent intensity. The labile iron pools in normal erythrocytes has been determined to be approximately 1 µM [18], [19]. Similar to deferiprone, the iron chelator FBS0701 at 100 µM was able to enter erythrocytes as evidenced by the increase in fluorescent intensity consistent with the release of iron from calcein (Figure 6A and B). Incubation of the FBS0701 and deferiprone for either one or two hours showed no difference in endpoint fluorescence intensity. To measure the removal of intracellular iron by chelation, erythrocytes at 4×106 cells/ml (0.125% hematocrit) were incubated for an hour with 100 µM of either deferiprone or FBS0701, washed and then placed in PBS overnight. Figure 6C shows that fluorescence in erythrocytes pre-treated with FBS0701 increased slightly from a mean fluorescence intensity (MFI) of 17 to 20. Pre-treatment with deferiprone showed a slight though statistically insignificant decrease in MFI. Addition of either iron chelators to cells previously incubated with FBS0701 showed a small rise of approximately 5 MFI compared to an increase of approximately 15 seen with either PBS or deferiprone-incubated cells demonstrating that in erythrocytes, FBS0701 depletes the labile pool of intracellular iron.
Discussion
A bioavailable oral iron chelator is an attractive and proven objective as an antimalarial agent. Much of the lack of efficacy of previous iron chelators for malaria therapy can be attributed to pharmacokinetic limitations including bioavailability, poor iron clearance efficiency and short-half life. FBS0701 has high solubility and a log p of −1.22 permitting good oral bioavailability and much better iron clearance than deferrioxamine and deferipone. Here we demonstrate activity against blood stage P. falciparum and also single dose cure in a lethal mouse malaria model. A potential limitation is the known interference to the artemisinin class of drugs. However, the evidence from this work indicates that FBS0701 may remove an irreplaceable source of iron in normocytes in mice. Previous studies on where iron is removed with deferoxamine in the calcein measurements showed a decrease in labile iron in both infected and uninfected erythrocytes. We have not demonstrated a decrease in nonheme iron in these studies but suggest that the effect we see in vivo is not from a new recompartmentalization of iron but from a decrease in erythrocyte levels. Consistent with other reports [9], [16], [21], we have demonstrated in vitro interference when both iron chelators and artemisinin are present simultaneously, but have not tested whether pre-treatment with FBS0701 before infection interferes with artemisinin activity.
Interestingly, the dose of iron chelator largely restricted lethal P. yoelii to young erythrocytes even almost 28 days after the drug was dosed and three weeks into infection. Mouse erythrocytes have a life span of about 60 days [25], [26]. Additional studies of the timing of iron chelator days to weeks before lethal P. yoelii infection will provide evidence to replenishment of this erythrocyte bioavailable source of iron for Plasmodium.
The mouse pharmacokinetic parameters are close to those obtained in the rat model [12]. Most of the iron-FBS0701 complex was excreted in the bile while free FBS0701 has renal excretion [12]. The relative level of FBS0701 in the rodent shows liver>kidney>plasma>pancreas>heart. Interestingly, humans show approximately ten times higher plasma concentrations compared to mouse for a given mg/kg dose. However, in studies comparing dose proportionality for cancer drugs where toxicity is a larger issue 100 mg/kg dose in mice represents a dose of 300 mg/m2 while a 10 mg/kg in humans is equivalent to 370 mg/m2 [27]. The implications are that in humans effective drug concentrations above minimal P. falciparum inhibition concentrations last less than ten hours. In the P. falciparum in vitro drug testing the drug is at continuous concentration for three days. In the mouse in vivo studies here we have demonstrated a persistent effect weeks after the drug was dosed. This data indicate that iron chelation is able to remove a bioavailable source which persists and plays a role in parasite inhibition. Unlike many of the long half-life blood stage active quinolines which rely on time above inhibition concentration to kill parasites, FBS0701 has an effect possibly by perturbing an iron compartment in the circulating erythrocytes.
The determination of relative labile iron concentrations using calcein indicates FBS0701 is able enter erythrocytes and bind iron bound to calcein. More importantly, in contrast to deferiprone, FBS0701 was able to remove labile iron from erythrocytes, an effect that persisted for at least 16 hours. This is the first study to demonstrate the egress of labile iron complexed with a chelator complex from erythrocytes. This has important clinical implications for the use of an iron chelator in the treatment of malaria as well as the ability of FBS0701 to remove iron from other cell types.
In the malaria iron chelator literature there is some debate whether iron chelators inhibit by limiting DNA replication which in many bacteria and mammalian cells is cytostatic or whether iron chelators are toxic and therefore parasiticidal to Plasmodium. Early stage gametocytes do not replicate DNA, so the data which show early stage inhibition with FBS0701 may indicate an additional toxic mechanism of inhibition at the high dose used in the gametocyte assay at the stages digesting the iron and hemoglobin rich erythrocyte cytoplasm.
In summary, FBS0701 demonstrates more potent inhibition for P. falciparum than previous iron chelators in clinical use. The activity persists in mice after the drug is below inhibition concentrations of approximately 5 uM. Our gametocyte data suggest a combined toxic mechanism in presence of erythrocyte cytosol ingestion in addition to the proposed mechanism of limiting DNA synthesis. In the lethal P. yoelii murine malaria model we show single oral dose cure. The removal of erythrocytic iron from intracellular bioavailable pools likely contributes to both the antimalarial activity and duration of effect of FBS0701 accounting for these effects in the relative absence of drug in plasma. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials after further preclinical testing to investigate liver stage activity, duration of blood stage interference with the artemisinins or possibly inhibition of transmission in mosquito stages ingesting FBS0701 exposed gametocytes.
Acknowledgments
We thank Graham Ellis for assistance with microscopy.
Footnotes
Competing Interests: Hugh Rienhoff is Founder, CEO, and shareholder in FerroKin BioSciences, Inc. FerroKin has licensed the patent to FBS0701. The patent holder is the University of Florida Research Foundation and the title of the patent is Desferrithiocin Polyether Analogues. The other authors do not have interests or affliations with FerroKin Biosciences or UFRF. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. FerroKin BioSciences did not provide financial support for this study.
Funding: The authors acknowledge partial support by the Bloomberg Foundation via the Johns Hopkins Malaria Research Institute and partial support by the National Institutes of Health 5U01 HD061241-03. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1.Sullivan DJ, Gluzman IY, Russell DG, Goldberg DE. On the molecular mechanism of chloroquine’s antimalarial action. Proc Natl Acad Sci U S A. 1996;93:11865–11870. doi: 10.1073/pnas.93.21.11865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.O’Neill PM, Posner GH. A medicinal chemistry perspective on artemisinin and related endoperoxides. J Med Chem. 2004;47:2945–2964. doi: 10.1021/jm030571c. [DOI] [PubMed] [Google Scholar]
- 3.Mabeza GF, Loyevsky M, Gordeuk VR, Weiss G. Iron chelation therapy for malaria: a review. Pharmacol Ther. 1999;81:53–75. doi: 10.1016/s0163-7258(98)00037-0. [DOI] [PubMed] [Google Scholar]
- 4.Gordeuk VR, Thuma PE, Brittenham GM, Zulu S, Simwanza G, et al. Iron chelation with desferrioxamine B in adults with asymptomatic Plasmodium falciparum parasitemia. Blood. 1992;79:308–312. [PubMed] [Google Scholar]
- 5.Gordeuk V, Thuma P, Brittenham G, McLaren C, Parry D, et al. Effect of iron chelation therapy on recovery from deep coma in children with cerebral malaria. N Engl J Med. 1992;327:1473–1477. doi: 10.1056/NEJM199211193272101. [DOI] [PubMed] [Google Scholar]
- 6.Thuma PE, Olivieri NF, Mabeza GF, Biemba G, Parry D, et al. Assessment of the effect of the oral iron chelator deferiprone on asymptomatic Plasmodium falciparum parasitemia in humans. Am J Trop Med Hyg. 1998;58:358–364. doi: 10.4269/ajtmh.1998.58.358. [DOI] [PubMed] [Google Scholar]
- 7.Portugal S, Carret C, Recker M, Armitage AE, Goncalves LA, et al. Host-mediated regulation of superinfection in malaria. Nat Med. 2011;17:732–737. doi: 10.1038/nm.2368. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Goma J, Renia L, Miltgen F, Mazier D. Iron overload increases hepatic development of Plasmodium yoelii in mice. Parasitology 112 ( Pt. 1996;2):165–168. doi: 10.1017/s0031182000084729. [DOI] [PubMed] [Google Scholar]
- 9.Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, et al. Artemisinins target the SERCA of Plasmodium falciparum. Nature. 2003;424:957–961. doi: 10.1038/nature01813. [DOI] [PubMed] [Google Scholar]
- 10.Wang J, Huang L, Li J, Fan Q, Long Y, et al. Artemisinin directly targets malarial mitochondria through its specific mitochondrial activation. PLoS One. 2010;5:e9582. doi: 10.1371/journal.pone.0009582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Bergeron RJ, Wiegand J, Bharti N, McManis JS, Singh S. Desferrithiocin analogue iron chelators: iron clearing efficiency, tissue distribution, and renal toxicity. Biometals. 2011;24:239–258. doi: 10.1007/s10534-010-9389-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Bergeron RJ, Wiegand J, McManis JS, Bharti N, Singh S. Design, synthesis, and testing of non-nephrotoxic desazadesferrithiocin polyether analogues. J Med Chem. 2008;51:3913–3923. doi: 10.1021/jm800154m. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Rienhoff HY, Viprakasit V, Tay L, Harmatz P, Vichinsky E, et al. A phase 1 dose-escalation study: safety, tolerability, and pharmacokinetics of FBS0701, a novel oral iron chelator for the treatment of transfusional iron overload. Haematologica. 2011;96:521–525. doi: 10.3324/haematol.2010.034405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bennett TN, Paguio M, Gligorijevic B, Seudieu C, Kosar AD, et al. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy. Antimicrob Agents Chemother. 2004;48:1807–1810. doi: 10.1128/AAC.48.5.1807-1810.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Elion GB, Singer S, Hitchings GH. Antagonists of nucleic acid derivatives. VIII. Synergism in combinations of biochemically related antimetabolites. J Biol Chem. 1954;208:477–488. [PubMed] [Google Scholar]
- 16.Bell A. Antimalarial drug synergism and antagonism: mechanistic and clinical significance. FEMS Microbiol Lett. 2005;253:171–184. doi: 10.1016/j.femsle.2005.09.035. [DOI] [PubMed] [Google Scholar]
- 17.Buchholz K, Burke TA, Williamson KC, Wiegand RC, Wirth DF, et al. A high-throughput screen targeting malaria transmission stages opens new avenues for drug development. J Infect Dis. 2011;203:1445–1453. doi: 10.1093/infdis/jir037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Darbari D, Loyevsky M, Gordeuk V, Kark JA, Castro O, et al. Fluorescence measurements of the labile iron pool of sickle erythrocytes. Blood. 2003;102:357–364. doi: 10.1182/blood-2002-03-0914. [DOI] [PubMed] [Google Scholar]
- 19.Loyevsky M, John C, Dickens B, Hu V, Miller JH, et al. Chelation of iron within the erythrocytic Plasmodium falciparum parasite by iron chelators. Mol Biochem Parasitol. 1999;101:43–59. doi: 10.1016/s0166-6851(99)00053-5. [DOI] [PubMed] [Google Scholar]
- 20.Prus E, Fibach E. The labile iron pool in human erythroid cells. Br J Haematol. 2008;142:301–307. doi: 10.1111/j.1365-2141.2008.07192.x. [DOI] [PubMed] [Google Scholar]
- 21.Meshnick SR, Yang YZ, Lima V, Kuypers F, Kamchonwongpaisan S, et al. Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu). Antimicrob Agents Chemother. 1993;37:1108–1114. doi: 10.1128/aac.37.5.1108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Petritus PM, Burns JM Suppression of lethal Plasmodium yoelii malaria following protective immunization requires antibody-, IL-4-, and IFN-gamma-dependent responses induced by vaccination and/or challenge infection. J Immunol. 2008;180:444–453. doi: 10.4049/jimmunol.180.1.444. [DOI] [PubMed] [Google Scholar]
- 23.Prus E, Fibach E. Flow cytometry measurement of the labile iron pool in human hematopoietic cells. Cytometry A. 2008;73:22–27. doi: 10.1002/cyto.a.20491. [DOI] [PubMed] [Google Scholar]
- 24.Shvartsman M, Fibach E, Cabantchik ZI. Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence. Biochem J. 2010;429:185–193. doi: 10.1042/BJ20100213. [DOI] [PubMed] [Google Scholar]
- 25.Horky J, Vacha J, Znojil V. Comparison of life span of erythrocytes in some inbred strains of mouse using 14C-labelled glycine. Physiol Bohemoslov. 1978;27:209–217. [PubMed] [Google Scholar]
- 26.Walker WS, Singer JA, Morrison M, Jackson CW. Preferential phagocytosis of in vivo aged murine red blood cells by a macrophage-like cell line. Br J Haematol. 1984;58:259–266. doi: 10.1111/j.1365-2141.1984.tb06084.x. [DOI] [PubMed] [Google Scholar]
- 27.Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep. 1966;50:219–244. [PubMed] [Google Scholar]