The Biodistribution of the Novel Heme and Hemoglobin Scavenger Apohemoglobin-Haptoglobin Complex in Healthy C57BL/6 Mice

Background: Apohemoglobin-haptoglobin (apoHb-Hp) complex represents a bioengineered complex designed to scavenge hemoglobin and heme. These substances are released into the intravascular space during hemolysis, leading to vasoconstriction, inflammation, and disease progression. The use of apoHb-Hp complex has already demonstrated promising results in sickle cell and beta-thalassemia mouse models, highlighting its potential as a therapeutic agent. However, there are still crucial aspects that remain poorly understood, particularly concerning the biodistribution and pharmacokinetics associated during chronic administration. Understanding how the drug is distributed throughout the body is essential for optimizing its therapeutic potential, ensuring patient safety, and guiding both drug development and clinical use.

Methods: To address these critical questions of apoHb-Hp biodistribution and pharmacokinetics, we conducted a study using twenty C57BL/6 mice from Jackson Laboratory. The mice were treated with the apoHb-Hp complex via daily injection every other day for different periods: 24 hours, one week, three weeks, and six weeks, with five mice in each subgroup. The dosage used was 80 mg/kg, and the concentration was 33.6 mg/mL. To evaluate the half-life and biodistribution of the complex, the last dose in each subgroup was radiolabeled with technetium-99m (99mTc-ApoHb-Hp).The intravascular 99mTc-ApoHb-Hp levels were measured after 30 minutes, 1 hour, 4 hours, and 24 hours to establish the drug's half-life. After the final tail vein sample collection, the mice were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were weighed and collected to determine the amount of 99mTc-ApoHb-Hp present in each organ.

Results: The results indicate that a significant portion of the 99mTc-ApoHb-Hp was accumulated in the liver, and regardless of the duration of dosing, the half-life for the 24 hour group was 10 hours, for the 1 week it was 6.5hrs, and for the 3 weeks it was 9hrs, showing no significant difference between the groups. hours. Liver accumulation favors the hepatocytes metabolism degradation of the apoHb-Hp complex from the body. There was no significant differences in half life based on dosing chronicity, likely suggesting that there was no additional degradation of ApoHb-Hp by immune mediated mechanisms such as antibody formation.

In conclusion, Biodistribution of ApoHb-Hp was predominantly in the liver in both acute setting ( single dose) and chronic setting ( multiple doses). The average half life of the infusion was 5 hours and the predominant clearance mechanism was likely via hepatic elimination. Further research and development may help improve our understanding of the biodistribution of the apo Hb- Hp complex, therapy opening new doors for the treatment of hemolytic anemias using the ApoHb-Hp complex.