Comparison of Two Animal Models for Evaluation of Defersirox Iron Chelation

Long-term storage iron (hemosiderin) is insoluble and not directly accessible to iron chelation. Ascorbate is a potent facilitator of redox cycling and facilitates the mobilization of cellular iron stores in patients chelated with deferoxamine. Ascorbate deficiency is quite common in thalassemia major and may create a phenotype of relative chelator refractoriness. However, the synergistic role of ascorbate supplementation has never been demonstrated for other chelators, including the oral chelator deferasirox. Ascorbate can be freely synthesized in many animals, making them unsuitable models to address this question. The osteodystrophic syndrome (ODS) rat is a spontaneous mutant lacking L-gulono-gamma-lactone oxidase, the initial enzyme in ascorbate synthesis. Iron loading and chelation have never been performed in ODS rats, so we report a pilot study aimed at optimizing organ iron loading and clearance.

Twenty-six 8-week old male ODS rats were maintained vitamin C replete by supplying ascorbate in the chow or drinking water. Fourteen animals were iron loaded using a combination of 0.4% TMH ferrocene and 3% carbonyl iron in standard rodent chow. Twelve animals were loaded with iron dextran (200 mg/kg/week) by subcutaneous injection. To characterize loading kinetics, two animals were analyzed at five, ten, and twelve weeks of oral iron loading as well as five and ten weeks of iron dextran injections. Sixteen animals were iron loaded for ten weeks (half enterally, half parenterally) and transitioned into the chelation arm. Iron chelation was performed with oral deferasirox at 75 mg/kg/dose, once daily five times per week. Chelation was administered for six and twelve weeks in six animals, divided equally between parenteral and enteral iron loading; sham chelation was given to the remaining four animals. Following euthanasia, H&E and iron staining were performed and tissue iron was quantified by atomic absorption.

Parenteral iron loading was well tolerated and yielded liver iron concentration (LIC) values of approximately 3 and 4.5 mg/g wet weight (∼11-16.8 mg/g dry weight) at five and ten weeks, respectively. Iron loading was predominantly reticuloendothelial, with little parenchymal redistribution. Spontaneous iron loss after 12 weeks of iron chelation was modest at 1.3% per week. In contrast, oral iron loading with the combination of TMH-ferrocene and carbonyl iron was poorly tolerated. Animals developed severe diarrhea and required fluid replacement and frequent dose reductions. These GI disturbances gradually lessened over a period of four weeks and animals received approximately 80% of the targeted iron dose. LIC values were lower at five weeks (2.2 mg/g ww) but nearly equivalent by 10 weeks (4.2 mg/g ww). Iron was entirely parenchymal, with little reticuloendothelial deposition. Spontaneous iron losses after 12 weeks of iron chelation were strikingly higher than for parenteral iron loading, measuring 4.7% per week.

Iron chelation with deferasirox was well tolerated in both groups. Iron chelation was less efficient in iron dextran loaded animals. LIC was 3.1 ± 0.7 mg/g ww compared with 3.8 ± 0.2 mg/g ww in sham chelated animals, p=0.25. In contrast, deferasirox treatment nearly completely cleared liver iron in TMH treated animals 0.4 ± 0.1 mg/g ww vs 1.5 ± 0.04 mg/g ww, p = 0.001, with most of the residual iron located primarily in reticuloendothelial store on histologic analysis.

Iron loading in the ODS rat can be performed with either iron dextran or TMH-ferrocene but the characteristics of iron staining, spontaneous iron loss, and chelator accessibility are completely different. Iron dextran loads the reticuloendothelial system. Iron redistribution to parenchymal tissues is sluggish, based upon the low spontaneous iron elimination rate and modest response to deferasirox therapy (1.5% per week). In contrast, TMH produced exclusively parenchymal loading, high spontaneous iron losses (4.7% per week) and more vigorous response to chelation (6.3% per week). Subsequent studies will determine whether the degree of ascorbate sufficiency modulates deferasirox efficacy in animals with primarily reticuloendothelial iron loading.

Nick:Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.