Heme Scavenging with Apohemoglobin-Haptoglobin Complex in Beta Thalassemia Intermedia Improves Anemia and Iron Indices: A Novel Therapeutic Approach

Beta-thalassemia intermediate (TI) is a genetic disorder of hemoglobin (Hb) synthesis characterized by anemia, ineffective erythropoiesis and iron overload. TI treatment is complex and regular transfusion therapy may be needed for growth failure, skeletal deformity, exercise intolerance, or when Hb levels decline because of progressive splenomegaly. Iron overload can be seen in TI, and serum ferritin measurement may often underestimate the extent of iron overload. There are limited therapeutic options for management of anemia and iron overload in TI. In this study, we look at the potential use of an engineered scavenger protein complex (apoHb-Hp) as a strategy to treat anemia in TI using a C57BL/6 mouse model.

Sixteen beta-thalassemic mice (C57BL/6 heterozygous for the Hbb β-globin gene deletion (Hbb ^td3th/BrjK) (beta-thalassemia, Jackson Laboratory)) were treated with 50 µL of apoHb-Hp complex at a concentration of 27.95 mg/mL for six weeks to simultaneously scavenge cell-free Hb and free heme. Animal weight and RBC parameters (Hb, hematocrit (Hct), red blood cell (RBC) count, red cell distribution width) were measured at baseline and at 6 weeks post treatment. Total iron levels, transferrin concentration and transferrin saturation were measured at the third and sixth week of treatment. At the end of the experiment, the spleen and liver weights were measured and markers of liver function (Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP)) were assessed. Further, total iron content of the liver and spleen was quantified using a using Perls' staining for non‐heme ferric iron (Fe3+).

The mean Hb at baseline for this cohort was 10.5 g/dL, Hct was 36% and RBC count was 7.6 10 ⁶/ µL. After treatment with apoHb-Hp complex for 6 weeks, Hb levels improved to 11.3 g/dL and Hct to 38% vs. Hb of 10.1 g/dL and Hct of 35% for vehicle treated animals. The mean total RBC count at was 9.2 10 ⁶/ µL following treatment with apoHb-Hp, and 7.7 10 ⁶/ µL for vehicle treated animals at 6 weeks. Iron parameters were also improved, with lower mean serum iron levels in the apoHb-Hp treated group at 6 weeks when compared to vehicle control (90.5 µg/dL vs. 135.2 µg/dL, p < 0.05). Serum transferrin levels were 131.1 mg/dL vs. 90.3 mg/dL (p<0.05) for the apoHb-Hp and vehicle treated groups at 6 weeks respectively. Furthermore, when compared to control mice, apoHb-Hp mice had a significant reduction in both liver and spleen weights at 6 weeks- liver 5.7g vs. 5.2g (p<0.05) and spleen 3.8g vs. 3.4g (p<0.05) respectively. Finally, liver function tests also showed improvement following treatment with apoHb-Hp at 6 weeks, ALT 71.1 units/L vs. 85.5 units/L for the vehicle, AST levels 180.2 units/L vs. 217.6 for the vehicle (p <0.05) of, and ALP levels 247.3 units/L vs. 304.4 units/L (p <0.05).

Our study demonstrates that apoHb-Hp can improve anemia and reduce iron toxicity in TI mice as well as improve liver and spleen parameters. Thus, apoHb-Hp may be a potential treatment strategy in this disease and merits further study.

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