Ceruloplasmin administration in the preclinical mouse model of aceruloplasminemia reveals a sex-related variation in biodistribution

Ceruloplasmin (CP) is an enzyme essential for maintaining the iron balance. When CP is absent due to a mutation in the CP gene, iron can’t be properly exported from cells, leading to iron accumulation in tissues. This leads to aceruloplasminemia (ACP), a rare genetic disorder, which results in iron overload and progressive neurodegeneration in the brain.

Mutations in the CP gene cause aceruloplasminemia (ACP), a rare genetic disorder characterized by abnormal accumulation of iron in various organs and progressive neurodegeneration in the brain. Previous studies have explored enzyme replacement therapy using purified CP to alleviate neurological symptoms in CP-deficient mice.

This study builds on that work by examining the sex-specific uptake and biodistribution of therapeutic CP across different organs in male and female CP-deficient mice (cpKO) using PET/CT imaging.

The distribution of radiolabeled 64Cu-CP in male and female cpKO mice at 6 and 10 months of age was investigated using PET/CT, with wild-type mice as controls.

Mice were injected with 64Cu-CP and imaged by PET/CT at 1, 3 and 21h. The 21h timepoint was selected to study sex or age-specific biodistribution of 64Cu-CP.

For PET imaging, mice were injected in the caudal vein with 5.12 ± 0.25 MBq of [64Cu]-CP and scanned at the different time points first by CT (X-CUBE) for 2 minutes and subsequently by PET (ß-CUBE) for 30 minutes. The data was reconstructed, co-registered, corrected for radioisotope decay, and expressed as Standardized Uptake Value (SUVmean) of the different organs.

The study found sex- and age-dependent differences in CP uptake in cpKO mice. At 6 months (early disease stage), male mice showed higher CP uptake than females, especially in the liver and cerebellum. However, by 10 months (advanced disease stage), this pattern reversed, with females displaying greater CP uptake than males.

These differences were not seen in wild-type mice, indicating that the effects are genotype-specific.

An unsupervised multivariate analysis showed that enzyme replacement therapy was more effective in male cpKO mice than in females, despite both sexes benefiting. This higher efficacy in males is likely due to their greater early uptake of CP at 6 months of age.

This study highlights a significant gap in preclinical ACP research, as most previous studies have either included only male CP-deficient mice or failed to report and analyze data by sex. By demonstrating distinct sex- and age-dependent differences in ceruloplasmin uptake, the findings underscore the importance of considering sex as a biological variable in both data analysis and experimental design.

a Imaging of [64Cu]-CP radiotracer injected in cpKO male and female mice of six months of age for kinetics of distribution determination. A representative image of a female (left) and a male (right) animal are shown for CT, PET and fused modality (from left to right) at the three different time points examined (from top to bottom). Scale bars = 1 cm. b PET kinetics in 6 months-old cpKO mice after the intravenous injection of [64Cu]-CP (N = 5; 3 females + 2 males); radiotracer distribution of males + females expressed as SUV (Mean ± SD). c radiotracer distribution of males + females expressed as tissue to background (muscle) (T/B) ratio (Mean ± SD).