Nanoparticle PET imaging of NPRC receptor in brown adipose tissue

Objectives: The recent discovery of functional brown adipose tissue (BAT) in adult humans has led to a marked interest in understanding its role in whole-body substrate metabolism and metabolic function. Currently, ¹⁸F-FDG is the standard for identifying BAT and quantifying its volume in people despite thelack of specificity and visualization of metabolically active BAT only. The natriuretic peptide receptor-C (NPR-C) is a clearance receptor for natriuretic peptides (NPs) and is much more abundant in human BAT than in white adipose tissue (WAT), making it a potential biomarker to determine BAT volume without the need for cold-induced metabolic stimulation. The goal of this study is to assess the up-regulation and imaging specificity of NPRC in BAT using a targeted CANF-Comb nanoprobe.

Methods: The NPRC targeted CANF-Comb nanoparticle was synthesized through controlled and modular chemistry and radiolabeled with ⁶⁴Cu (⁶⁴Cu-CANF-Comb) for PET imaging. Biodistribution was performed in wildtype C57BL/6 mice. PET imaging of BAT was carried out in both wildtype C57BL/6 mice and DB/DB obese, dyslipidemic mice at room temperature. The non-targeted Comb (⁶⁴Cu-Comb) was used as a negative control in both models. The PET imaging specificity was further confirmed by competitive receptor blocking studies. Immunohistochemistry was performed on mouse BAT and WAT to verify the presence of NPRC receptor.

Results: The ⁶⁴Cu-CANF-Comb was prepared with high specific activity, enabling tracer amount administration for PET imaging. Biodistribution showed extended blood circulation and moderate liver accumulation. Compared to the weak accumulation of ⁶⁴Cu-Comb in BAT in both WT and DB/DB mice, the targeted ⁶⁴Cu-CANF-Comb showed significantly increased uptake in BAT in both WT (SUV=0.457± 0.05, n=4, p< 0.005) and DB/DB mice (SUV=0.315 ± 0.02, n=4, p<0.005). With competitive blocking, the uptake of ⁶⁴Cu-CANF-Comb in DB/DB mice was significantly (p<0.01, n=4) reduced to a level (0.166 ± 0.04, n=4) similar to ⁶⁴Cu-Comb (SUV=0.188 ± 0.02, n=4), indicating the NPRC targeting specificity of ⁶⁴Cu-CANF-Comb in BAT. Of note, uptake of ⁶⁴Cu-CANF-Comb in WAT present in the gonadal regions of both types of mice was statistically (p<0.005, n=4) lower than ⁶⁴Cu-CANF-Comb uptake in BAT. In BAT tissues of both C57BL/6 and DB/DB mice, the over-expression of NPRC was verified by immunohistochemistry (IHC) while only minimal NPRC expression was observed in WAT, which validated the PET imaging data. In addition, anecdotally, whole body ⁶⁴Cu-CANF-Comb studies that we performed in healthy human volunteers for biodistribution assessment at thermoneutral conditions, showed, that ⁶⁴Cu-CANF-Comb uptake in the supraclavicular regions in at least 2 subjects (SUVs=1.29-1.42) was 10-fold higher than uptake in chest wall subcutaneous WAT (0.13 to 0.19). Conclusion: We have identified specific up-regulation of NPRC receptor in both mouse and human BAT at thermoneutral conditions with an insignificant level of expression of NPRC in WAT. The targeted ⁶⁴Cu-CANF-Comb demonstrated NPRC receptor mediated uptake in BAT, suggesting that ⁶⁴Cu-CANF-Comb may be a potential PET imaging biomarker for the identification of BAT volume without the need for cold-induced metabolic stimulation in both pre-clinical studies and human trials.