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Thiazolidinones reduce iron overload
alanine aminotransferase and lactate dehydrogenase did not exhibit significant changes (P>0.05) (Online Supplementary Figure S13).
We then extended the animal studies for a longer time period, 4 weeks. As shown in Online Supplementary Figure S14A, serum hepcidin concentration was significantly increased by 2.5 fold in Hfe-/- mice after administration of compound 93 at a dose of 10 mg/kg body weight for 4 weeks, compared to the level in untreated Hfe-/- mice (P<0.05). In response to the change in serum hepcidin con- centration, serum iron and hepatic iron were reduced by ~20% and ~30%, respectively (P<0.05) (Online Supplementary Figure S14B,C). As a consequence, splenic iron content was increased by ~40% in Hfe-/- mice after treatment with compound 93 relative to the iron content in untreated controls (P<0.05) (Online Supplementary Figure S14D). Tissue iron staining further supported the results of the liver and splenic iron measurements (Online Supplementary Figure S14E). Additionally, no significant increases of serum interleukin-6, aspartate aminotrans- ferase, or alanine aminotransferase levels were found in mice after 4 weeks of treatment with compound 93, ruling out the occurrence of inflammation and hepatic injury (Online Supplementary Figure S15).
To model the condition of patients undergoing iron removal, we fed 9-week old male Hfe-/- mice with a low iron diet (4 ppm) for 3 weeks. Immediately after this pre- treatment, these Hfe-/- mice on a normal diet were treated with compounds 93, 156 and 165 at a dose of 30 mg/kg body weight every 3 days for 2 weeks (experimental scheme in Figure 6A). After treatment with compounds 93, 156 and 165, the serum hepcidin concentration was increased by approximately 2 fold, relative to that of untreated Hfe-/- mice (P<0.05) (Figure 6B). Consequently, serum iron and transferrin saturation were significantly reduced (P<0.05) (Figure 6C and Online Supplementary Figure S16A), and serum transferrin and splenic iron were increased (P<0.05) (Online Supplementary Figure S16B and Figure 6D), associated with decreased hepatic iron con- tent (P<0.05) (Figure 6E). The tissue iron changes were further confirmed by iron staining, and we also noted increased iron concentration in the duodenum, indicative of inhibition of the transfer of absorbed iron into plasma (Figure 6F). Serum interleukin-6, aspartate aminotrans- ferase and alanine aminotransferase were not significant- ly altered in Hfe-/- mice by the treatments (Online Supplementary Figure S17).
in the liver and spleen was reduced by >30% and 35%- ~50%, respectively (P<0.05) (Figure 7D,E), indicating an effective relief of iron overload in Hbbth3/+ mice by these compounds. Spleen and liver iron staining supported these results (Figure 7F).
Compounds 93, 156 and 165 ameliorated iron overload and mitigated ineffective erythropoiesis in Hbbth3/+ mice
In β-thalassemia, deficiency of β-globin causes an imbalance between a- and β-globin, so that excess a-glo- bin tetramers aggregate in erythroblasts, leading to apop- tosis of orthochromatic erythroblasts and reactive increases in earlier erythroblast forms. Thus, β-tha- lassemia syndromes often manifest with severe anemia with ineffective erythropoiesis. To understand the effect of our compounds on erythroblast maturation, we exam- ined erythropoiesis in Hbbth3/+ mice. As shown in Figure 8A, the hemoglobin level was elevated by ~15% by these compounds (P<0.05). Additionally, red blood cell content was increased ~10% upon treatment with the com- pounds compared to the erythrocyte content in untreated controls (P<0.05) (Figure 8B). Furthermore, blood smears revealed an increased number of red blood cells with nor- mal morphology and a decrease of damaged or deformed erythrocytes in treated mice (Figure 8C, denoted by arrows). To corroborate these findings, flow cytometry analysis was performed to define erythroid populations using the erythroid markers TER119 and CD44 and cellu- lar size. As shown in Figure 8D, compounds 93, 156 and 165 increased the percentage of the P7 subpopulation (indicative of mature red blood cells) in bone marrow by 1.2 fold, 1.1 fold and 1.2 fold, respectively, with corre- sponding declines in the proportions of P6 subpopula- tions, compared to those in untreated controls. Similar findings were observed for erythropoiesis in spleens from treated mice, as these compounds increased the percent- age of the P7 subpopulation in the spleen by 1.4 fold, 1.5 fold and 1.3 fold, respectively, with corresponding declines in the proportions of P5 subpopulations, com- pared to those in untreated controls (Online Supplementary Figure S18).
In addition, we explored the consequences of a longer treatment, for 4 weeks. As shown in Online Supplementary Figure S19A, administration of compound 93 resulted in an approximately 2-fold increase of serum hepcidin in Hbbth3/+ mice relative to the level in untreated Hbbth3/+ mice (P<0.05). As a result, serum iron, hepatic iron and splenic iron were significantly reduced by 20-25% (P<0.05) (Online Supplementary Figure S19B-D). Iron staining sup- ported the changes found in liver and spleen iron (Online Supplementary Figure S19E). There was also a significant reduction of spleen size and weight in Hbbth3/+ mice treat- ed with compound 93 (P<0.05) (Online Supplementary Figure S19F,G), suggestive of an improvement in the pre- viously ineffective erythropoiesis. Furthermore, increases in hemoglobin concentration and red blood cell count were found in Hbbth3/+ mice 4 weeks after administration of compound 93, in comparison to those in untreated Hbbth3/+ mice (P<0.05) (Online Supplementary Figure S20A,B). The findings of blood smears and analysis of representative erythroid populations of bone marrow and spleen were also consistent with the increasing concen- tration of hemoglobin and red blood cell content (Online Supplementary Figure S20C,D). Additionally, we measured the concentration of malondialdehyde, which is indica- tive of oxidative stress damage, such as injury by reactive oxygen species,39,40 and of erythropoietin. As show in Online Supplementary Figure S21, the malondialdehyde
Even untransfused patients with β-thalassemia suffer from iron overload and associated organ damage due to inappropriately low production of hepcidin. We therefore tested our compounds in a mouse model of thalassemia intermedia, Hbbth3/+ mice,38 given a 2-week course of treat- ment. Serum hepcidin was significantly increased by ~45% in Hbbth3/+ mice treated with compounds 93, 156 and 165, relative to the levels in untreated mice (P<0.05) (Figure 7A). As a consequence, the serum iron concentra- tion dropped by ~30% (P<0.05) (Figure 7B). Similarly to what occurred in Hfe-/- mice, the serum ferritin concentra- tion dropped ~28% (P<0.05) (Figure 7C), indicating that treatment with these agonists greatly diminished hyper- ferritinemia in Hfe-/- and Hbbth3/+ mice, and the iron content
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