The iron chelator deferasirox protects mice from mucormycosis through iron starvation

Abstract

Mucormycosis causes mortality in at least 50% of cases despite current first-line therapies. Clinical and animal data indicate that the presence of elevated available serum iron predisposes the host to mucormycosis. Here we demonstrate that deferasirox, an iron chelator recently approved for use in humans by the US FDA, is a highly effective treatment for mucormycosis. Deferasirox effectively chelated iron from Rhizopus oryzae and demonstrated cidal activity in vitro against 28 of 29 clinical isolates of Mucorales at concentrations well below clinically achievable serum levels. When administered to diabetic ketoacidotic or neutropenic mice with mucormycosis, deferasirox significantly improved survival and decreased tissue fungal burden, with an efficacy similar to that of liposomal amphotericin B. Deferasirox treatment also enhanced the host inflammatory response to mucormycosis. Most importantly, deferasirox synergistically improved survival and reduced tissue fungal burden when combined with liposomal amphotericin B. These data support clinical investigation of adjunctive deferasirox therapy to improve the poor outcomes of mucormycosis with current therapy. As iron availability is integral to the pathogenesis of other infections (e.g., tuberculosis, malaria), broader investigation of deferasirox as an antiinfective treatment is warranted.

Figure 1. Deferasirox induces the expression of rFTR1.

(A) RT-PCR expression of rFTR1 from R. oryzae mycelia incubated in iron-replete, iron chelation (deferasirox), or reversal of iron chelation (deferasirox saturated with ferric chloride) conditions. Expression of the 18S rDNA was included to verify the quality of RNA extraction. (B) Diagram demonstrating the strategy for constructing R. oryzae GFP expression vector. Promoter denotes either rFTR1p or ACT1p. (C) GFP expression in R. oryzae driven by rFTR1p or ACT1p as determined by confocal images and flow cytometry of R. oryzae grown in iron-replete medium or medium containing deferasirox alone or deferasirox saturated with ferric chloride. GFP expression was revealed by green fluorescent cells by confocal microscopy, and the percentage of fluorescent cells in channel FL1 (y axis) by flow cytometry. In contrast to GFP under the control of the ACT1p, which was constitutively expressed regardless of growth conditions, GFP under the control of the rFTR1p was expressed only in the presence of iron chelation conditions (deferasirox [Def]).

Figure 2. Deferasirox protects diabetic ketoacidotic mice from hematogenously disseminated R. oryzae infection.

Mice infected via the tail vein were treated with placebo (hydroxypropylcellulose carrier), deferasirox, or deferasirox plus iron (FeCl₃, 10 mg/kg) to reverse the effect of iron chelation. (A) Survival of mice (n ≥ 7 per group) infected with R. oryzae 99-892 (2.2 × 10⁴ spores) and treated with various doses of deferasirox. (B) Brain and kidney fungal burden of mice (n = 11 per group) infected with R. oryzae 99-892 (4.2 × 10⁴ spores) and treated with placebo, deferasirox (10 mg/kg twice daily), or deferasirox plus iron. Organs were harvested on day 4 after mice received 3 daily treatments. Data are displayed as median ± interquartile ranges. The y axis reflects the lower limit of detection of the assay. (C) H&E-stained kidney sections of mice infected with R. oryzae 99-892 and treated with deferasirox, deferasirox plus ferric chloride, or placebo as described in B. Arrows indicate R. oryzae hyphae in tissue. Sections are representative of findings throughout organs in all mice in the respective groups. Original magnification, ×400. (D) Survival of diabetic ketoacidotic mice (n = 24 from 3 separate experiments with similar results) infected via the tail vein with R. oryzae 99-880 (average inoculum, 1.3 × 10³ spores) and 24 hours later treated with 10 mg/kg deferasirox twice daily for 7 days. *P < 0.05 for survival and ^†P < 0.002 for tissue fungal burden compared with placebo or deferasirox plus ferric chloride.

Figure 3. Deferasirox protects diabetic ketoacidotic mice infected intranasally with R. oryzae.

Survival of mice infected with 10⁷ spores of R. oryzae 99-880 and 24 hours later treated with placebo (hydroxypropylcellulose carrier, n = 13), deferasirox (10 mg/kg, twice daily, n = 13), or deferoxamine (50 mg/kg, n = 8). *P < 0.009 compared with placebo- or deferoxamine-treated mice; **P = 0.047 compared with placebo-treated mice.

Figure 4. Iron chelation increases splenic Th1 and Th2 lymphocyte frequencies and increases the levels of proinflammatory cytokines compared with iron overload.

Diabetic ketoacidotic mice were infected via the tail vein with 3.1 × 10⁴ spores of R. oryzae 99-892 and 24 hours later treated with placebo, deferasirox, or deferasirox plus ferric chloride. Mice (n = 11) were sacrificed and spleens and kidneys collected 4 days after infection. Data are presented as median ± interquartile ranges. (A) Frequencies of Th1 and Th2 splenocyte. (B) Whole-organ cytokine analysis by cytometric bead array. *P < 0.02 compared with placebo or deferasirox plus ferric chloride; **P < 0.05 and ^†P < 0.07 compared to deferasirox plus ferric chloride.

Figure 5. Deferasirox therapy acts synergistically with LAmB.

(A) Survival of diabetic ketoacidotic mice (n > 16 from 2 separate experiments with similar results) infected via the tail vein with R. oryzae 99-880 (average inoculum, 1.5 × 10³ spores) and treated with combination of deferasirox (10 mg/kg twice daily for 7 days) and LAmB (15 mg/kg for 4 days). (B) Tissue R. oryzae burden in brains and kidneys of mice (n > 7) infected via the tail vein with R. oryzae 99-880. Treatment began 24 hours after infection and consisted of placebo, deferasirox (10 mg/kg, twice daily), LAmB (15 mg/kg/d), or a combination of both drugs. Organs were harvested on day 3 after animals received 2 daily treatments. Data are presented as median ± interquartile ranges. The y axis reflects the lower limit of detection of the assay. *P < 0.003 compared with placebo; **P < 0.003 compared with placebo, deferasirox, or LAmB; ^†P < 0.01 compared with placebo or deferasirox.

Figure 6. Deferasirox is effective in treating R. oryzae infections in neutropenic mice.

Cyclophosphamide-treated mice (n = 19 from 2 separate experiments with similar results) were infected via the tail vein with 2.7 × 10³ spores of R. oryzae 99-892. Mice were treated 24 hours after infection with placebo or deferasirox (10 mg/kg) administered every day or every other day for a total of 5 doses. *P = 0.037 compared with placebo.