Treatment of Mucormycosis with Deferasirox in the Laboratory and in a Clinical Case
Background
Mucormycosis is a fungal infection with an extremely high mortality rate (over 50%), with patients often succumbing despite the use of modern state-of-the-art treatments. For particular patient populations, including those with central nervous system involvement, mortality rises to 80%-100% (1). Fungi of the order Mucorales, ubiquitous in the environment, cause this disease. These fungi cause mucormycosis by invading the host organism primarily through inhalation of conidia, ingestion, or infection of open wounds (2). Rhizopus oryzae is the most common cause of mucormycosis (1). Mucormycosis generally infects immunocompromised individuals. Diabetic ketoacidosis and neutropenia are risk factors because these patients become immunocompromised as a result of their conditions (2).
It has previously been noted that many microbial pathogens require iron. In fact, infected mammals often use iron sequestration as a defense mechanism. It follows, therefore, that the use of iron chelating drugs may be an effective treatment for many pathogens (1). Mucormycosis has also been shown to require iron to grow in the host (3). However, this possible treatment has long been ignored because of conflicting data from previous trials with the iron chelating drug deferoxamine. Deferoxamine could successfully sequester the iron available in the host serum, but the risk of developing mucormycosis for animals treated with deferoxamine would paradoxically increase. Eventually, it was found that Mucorales fungi were able to specifically bind deferoxamine and remove the sequestered iron. Deferoxamine therefore facilitated the iron uptake of Mucorales (1).
The approval of the new oral iron chelating drug deferasirox by the United States Food and Drug Administration again brought forth the possibility of treating microbial pathogens, like those causing mucormycosis, with iron chelation. As a result, a collection of researchers at the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, the UCLA David Geffen School of Medicine, Charles River Laboratories, and the National Center for Agricultural Utilization Research conducted research and produced a paper detailing the effectiveness of deferasirox on mucormycosis in murine animal models. Consideration of this paper, “The iron chelator deferasirox protects mice from mucormycosis through iron starvation” (1) along with a clinical case, “Deferasirox, an iron-chelating agent, as salvage therapy for rhinocerebral mucormycosis” (3) reveals the promising possibility of the future use of iron chelation as a novel treatment for mucormycosis.
Results
The initial step in the investigation was to confirm deferasirox’s iron chelation ability. One R. oryzae gene, rFTR1, was known to show upregulated expression in response to iron starvation. The authors therefore decided to use rFTR1 to indicate a state of iron starvation. R. oryzae strain 99-880, a clinical brain isolate, was tested in three different environments: “iron replete”, “iron depleted”, and a control. “Iron replete” was a growth medium with additional 350 μM ferric chloride, whereas “iron depleted” added 350 μM ferric chloride and 2 mM deferasirox to sequester the iron, and the control added 2 mM deferasirox with 6 mM ferric chloride to render the deferasirox ineffective. RT-PCR amplification of the extracted RNA showed rFTR1 expression only in the “iron depleted” scenario. 18S rDNA was used to provide a baseline expression level for comparison and to show that there was no genomic DNA contamination (1).
Two DNA vectors were constructed for the purpose of visualizing rFTR1 expression in vivo. The GFP gene was placed behind either the rFTR1 promoter or the actin gene ACT1 promoter. Both versions included pyrF, required for uracil synthesis, and were placed in the M16 strain, which was mutagenized from 99-880 to be pyrF-null. This allowed the use of a selection system to create a population of M16 cells that would express GFP in response to the upregulation of rFTR1 or ACT1 expression. These cells were grown in the same control, “iron replete” and “iron deficient” media previously used. The M16 cells with rFTR1 promoter vectors showed GFP expression only in the “iron deficient” scenario, whereas those with the ACT1 promoter vectors showed GFP expression regardless of the scenario, confirming the effectiveness of the vectors. Flow cytometry was then used to confirm and quantify the GFP fluorescence of the M16 cell cultures (1).
A quick test was then performed on 29 Mucorales organisms to determine susceptibility to deferasirox.Deferasirox was determined to be fungicidal against nearly all the tested organisms (the only exception was R. oryzae type I NRRL13440). Deferasirox was considered fungicidal if the minimum fungicidal concentration was at most 4 times the minimum inhibitory concentration required to inhibit 90% of the isolates. Even at low deferasirox concentrations, fungicidal effects were noted within 24 hours, suggesting a time dependent effect rather than a concentration dependent effect. A short test was then performed by growing R. oryzae, adding deferasirox, and then adding iron to half of the samples. The iron addition reversed the deferasirox’s cidal effects, and even supported growth, but this data was not included in the paper (1).
Having demonstrated the ability of deferasirox to induce iron starvation to a fungicidal point, the next step was to demonstrate deferasirox’s effects in vivo with a murine model. Diabetic ketoacidosis was induced in BALB/c male mice with 210 mg/kg streptozotocin to destroy the pancreas’s insulin producing β cells. R. oryzae strain 99-892, a clinical pulmonary isolate, was used to simulate disseminated infection by injection of 2.2 x 104 spores into the tail vein. The experimental mouse cohorts were treated with 1, 3, or 10 mg/kg of deferasirox. Control mice were treated with a placebo, deferasirox with excess ferric chloride, or ferric chloride alone, but this data was not shown. Deferasirox was shown to improve survival markedly, with some suggestion of a minor dose dependent response, but this observation was not followed up on. More mice were tested and colony forming units were counted in the kidneys and brain, and deferasirox was shown to also significantly reduce the fungal burden. More mice were infected with 4.2 x 104 spores of 99-892. Histology with hemotoxylin and eosin staining showed that hyphae failed to develop in samples from mice that underwent deferasirox treatment. Improved survival with deferasirox treatment also resulted when testing was performed with 1.3 x 103 spores of the more virulent 99-880 strain (1).
To simulate the usual pulmonary route of infection, diabetic ketoacidotic mice were infected intranasally with 107 spores of 99-880 to deliver a median 6.5 x 104 spores to the lungs. Mice were treated with a placebo, deferasirox, or deferoxamine. Deferoxamine was shown to worsen survival rates compared to the placebo, whereas deferasirox improved survival rates (1).
The authors then surmised from their observations that excess iron could directly suppress the host inflammatory response. Helper T cells Th1 and Th2 levels as well as levels of inflammatory cytokines TNF and IFN-γ were tested in diabetic ketoacidotic mice infected with 3.1 x 104 spores of 99-892. Treatment with deferasirox resulted in a rise in splenic Th1 and Th2 levels over levels observed in mice treated with either placebo or deferasirox and excess iron. The deferasirox regimen also resulted in mice with significantly increased TNF and IFN-γ levels in the spleen, as well as increased IFN-γ levels in the kidney. No effects were noted on Treg cells or on lymphocyte apoptosis frequency (1).
Since one current front-line treatment for mucormycosis is amphotericin B, deferasirox was tested alongside amphotericin B treatment to determine the efficacy of this combination. Again, diabetic ketoacidotic mice were infected with 99-880 through the tail vein. The mice were treated with either a placebo, liposomal amphotericin B (LAmB), deferasirox, or both LAmB and deferasirox. This series of experiments showed a surprising synergistic effect in the combination of LAmB and deferasirox that drastically improved survival rates even over the improved survival rates of either LAmB or deferasirox alone. The tissue burden in both the brain and the kidneys were also drastically reduced by the combination of LAmB and deferasirox, whereas LAmB was able to significantly reduce kidney tissue burden only (1).
Finally, the effect of deferasirox in a neutropenic murine model was tested. BALB/c mice were “myeloablated with cyclophosphamide” (1) at a dosage of 200 mg/kg, removing the ability to produce immune cells like neutrophils. These mice were infected with 2.7 x 103 spores of 99-892. Oddly, this study showed that treatment every other day with deferasirox was much more effective than daily treatment. Theorizing that a toxicity effect may be the root of this phenomenon, the authors performed toxicity tests, but found no evidence of deferasirox toxicity (1).
The clinical study involved a 40 year old male presenting with diabetic ketoacidosis, pain in the left retrobulbar area, and palsy in the left cranial nerve VI (3). The patient was therefore at extreme risk with diabetic ketoacidosis as well as central nervous system involvement. Developments in the left eye included paralysis of the extraocular muscles responsible for eye movement, and increasing pressure causing the eye to protrude outward (3), known as “proptosis” (4). The patient began receiving amphotericin B. A few weeks later, the patient received surgery to remove the contents of the left orbital area, which revealed an extensive ischemic area with much necrosis in the extraorbital muscles. LAmB treatment quickly followed, along with caspofungin. A postoperative MRI showed heavy clotting in the left cavernous sinus (3), common for rhinocerebral mucormycosis infections (4).
Follow up MRIs revealed continuing mucormycosis progression to involve cranial nerve V and the development of a new brain lesion in the pons and left cerebellum. Renal insufficiency developed, requiring a dilution of LAmB dosage. Meanwhile, subsequent MRIs exposed the continued growth of the mucormycosis infection. Salvage therapy was attempted with a seven day course of deferasirox (3). At this point, the synergistic relationship of LAmB and deferasirox must have come into play. The patient improved rapidly, and a final MRI showed significant improvement to the point where LAmB dosing was discontinued after the deferasirox therapy was completed. Four months later, the patient was asymptomatic, with no worsening neurological effects, and stabilization with no apparent changes noticed in subsequent MRIs (3).
Discussion
The paper and clinical case in conjunction provide evidence that the iron chelating drug deferasirox can successfully sequester iron and starve R. oryzae to treat mucormycosis. The laboratory testing first confirmed deferasirox’s ability to sequester iron and stress R. oryzae through iron starvation. Next, deferasirox was confirmed to have fungicidal activity against various Mucorales fungi, and it was demonstrated that this activity was based on iron starvation. Mice with diabetic ketoacidosis were tested as a model for humans with the same risk factor. Deferasirox was shown to be able to reduce fungal burden, reduce R. oryzae hyphae formation in tissue, and improve survival for infected mice. Improved survival was shown for both disseminated infection and the more common intranasal infection. The paper then revealed conflicting data regarding iron availability and the immune response. Deferasirox iron chelation raised Th1 and Th2 levels as well as TNF and IFN- γ levels in the spleen. Deferasirox also raised IFN-γ in the kidneys. Finally, deferasirox showed a synergistic effect with LAmB in the treatment of diabetic ketoacidotic mice. Deferasirox in combination with LAmB also more successfully reduced fungal burden in the brain and kidneys. Finally, deferasirox was shown effectively increase survival rates of neutropenic mice, although not as effectively as with diabetic ketoacidotic mice, and optimally with treatment every other day. Treatment in combination with LAmB was not tested (1). It appears clear that at least diabetic ketoacidotic patients with mucormycosis infection by R. oryzae should be able to be treated with some success with deferasirox.
It would have been interesting to investigate the dose response of deferasirox treatment on R. oryzae strains 99-892 and 99-880 to determine why a dose response trend in survival was noted when testing diabetic ketoacidotic mice infected with 99-892. The comparison of the two strains in a dose response test may have revealed some interesting responses between the more virulent strain and 99-892. Further testing with similarly infected diabetic ketoacidotic mice could also have helped determine an optimal treatment concentration for 99-892.
The clinical case study provided anecdotal evidence that deferasirox can successfully act as salvage therapy for mucormycosis. Since the patient embodied many of the characteristics of mucormycosis sufferers, including diabetic ketoacidosis, central nervous system and cranial nerve involvement, and facial sinus infection extending into the brain, this case is a promising example (2, 3). Further study is clearly needed before deferasirox can be widely employed against microbial infections, but this is a positive first step.
Obvious future directions for further research are brought to mind by this research. Due to the sudden increase in the amount of data withheld during the immune response testing, it appears that the authors are already considering further research into effects of the iron chelator on the host immune response. This testing should aim to explain the elevated TNF and IFN-γ levels when deferasirox was added, as well as the increased Th1 and Th2 splenocyte levels and the unexplained Th1 cytokine and Th2 cytokine levels (1). The authors suggest that the effect of iron on host immune response could vary from host to host. This assertion was based on conflicting findings between their own data and other studies, sometimes involving iron chelators other than deferasirox (1). Due to the involvement of other drugs, the investigation of this matter may need to become more complex. However, it is still important for this matter to be pursued further in order to concretely establish iron’s effects on the host immune response.
The ultimate goal of future research should be to better establish the parameters for clinical use of deferasirox against mucormycosis. Perhaps if this future research determines that iron can directly suppress the host’s inflammatory response, then clinical treatment with deferasirox would increase the inflammation in response to mucormycosis while helping to clear the infection simultaneously. This combined effect may be more successful at fighting mucormycosis than either alone. On the other hand, if iron has the opposite effect on the immune response, then deferasirox treatment would relieve inflammation while killing the fungal infection. This relief may reduce the body’s ability to deal with the mucormycosis, and therefore more deferasirox may be necessary to fully clear the infection. Either way, detailed knowledge of the effects of deferasirox treatment will be important for clinical use. Additionally, it is important that the optimal treatment regimen be determined, whether that is the daily treatment with deferasirox, treatment every other day with deferasirox, treatment in conjunction with LAmB, or some other combination of factors. Furthermore, as the authors suggested, better toxicity data for deferasirox should be determined, since the effect of toxicity in clinical deferasirox applications at this time is still unclear (1).
Lastly, since iron chelation holds such promise for widespread application across many different microbial infections, the authors suggest future research into the applicability of iron chelators, and deferasirox in particular, as a future therapy against other important pathogens. The availability of an iron supply is important to many microbial pathogens, and if iron chelation and deferasirox treatment can be found to reduce the iron supply and starve microbes, iron chelation as a clinical therapy may find applications across many fields (1).
References
1. A. S. Ibraham, et al., The Journal of Clinical Investigation 117(9), 2649 (2007).
2. N. F. Crum-Cianflone and D. Eisen, Mucormycosis (2006). Available at http://www.emedicine.com/MED/topic1513.htm (04 March 2008).
3. C. Reed, et al., Antimicrobial Agents and Chemotherapy 50(11), 3968 (2006).
4. D. S. Smith, Mucormycosis (2006). Available at http://www.nlm.nih.gov/medlineplus/ency/article/000649.htm (04 March 2008).