Evaluation of new deferasirox-derived compounds for the treatment of iron overload

Abstract

Abstract Introduction: Iron overload is a major complication in individuals requiring frequent blood transfusions or those with genetic iron disorders. Because the body lacks a natural mechanism for iron excretion, iron chelation therapy is essential. Although three chelating agents are available, their use is limited by the need for high doses to achieve efficacy and by adverse effects. Combination therapies are often more effective than monotherapies, but pharmacokinetic and pharmacodynamic limitations of the current drugs highlights the need for improved alternatives to the already existing alternatives. In this context, we evaluated newly developed analogues of deferasirox (DFX) for their ability to chelate both free and intracellular iron, as well as their effects in iron-overloaded C57BL/6 mice.Methods: Chelation efficiency was assessed using calcein fluorescence, which decreases when iron binds and is restored upon iron removal by chelators. Free iron binding was first evaluated by adding DFX or one of the new compounds (C1 and C4; 0.05–5µM) to a solution containing calcein and ferrous diammonium sulfate. Fluorescence recovery was measured every 4 minutes for 2 hours in a microplate spectrophotometer. For intracellular iron chelation, H9c2 cells were loaded with ferric ammonium citrate for 24 hours, incubated with calcein-AM for 30 minutes, and treated with chelators. Fluorescence was measured every 8 seconds for 2 minutes. In vivo, 4-week-old male C57BL/6 mice were fed a 0.2% ferrocene diet for 14 weeks and then treated via gavage with vehicle, DFX, C1, or C4 for 2 weeks. Serum ferritin levels were quantified by ELISA, and iron deposition in the liver, spleen, heart, and aorta were evaluated by Prussian blue staining. Image analysis was performed in ImageJ to quantify hemosiderin.Results: In solution, calcein fluorescence recovery was concentration-dependent, indicating iron chelation. At higher concentrations, C1 exhibited rapid chelation comparable to DFX, peaking at 10 minutes, whereas C4 displayed slower activity, with fluorescence still increasing beyond 20 minutes. Both compounds were cell-permeable and restored fluorescence in H9c2 cells, confirming intracellular iron chelation. All chelators reached a plateau within 15 seconds, suggesting rapid internalization and action. At 1µM, C1 reached 118% of DFX activity, and C4 reached 108%. After confirming activity in vitro, we next assessed whether these compounds could reduce systemic and organ iron levels in iron-overloaded mice. Preliminary in vivo results after 2 weeks of treatment showed a trend toward reduced serum ferritin in the C4 group compared with untreated mice (20,752 ± 221.7 vs. 27,664 ± 4,244 ng/ml, respectively), contrasting with no significant reduction observed with C1 and DFX in this short period. Although the effect wasn't statistically significant, treatment with C4 acted reducing liver hemosiderin deposit (78.8 ± 14.2 vs. 64.5 ± 4.2 µm2), while DFX reduced the deposition on the spleen (32.2 ± 8,7 vs. 24.3 ± 3.6 µm2), when compared to untreated and C1 mice.Conclusion: The new DFX analogues effectively chelated both free and intracellular iron, restoring calcein fluorescence. In solution, DFX and C1 acted more rapidly than C4, but both C1 and C4 were more efficient than DFX in chelating intracellular iron. Despite the short treatment duration, C4 showed a tendency to reduce serum ferritin, suggesting in vivo activity. Ongoing studies with prolonged treatment will further elucidate the therapeutic potential of C1 and C4.