64Cu-DOTHA2-PSMA, a Novel PSMA PET Radiotracer for Prostate Cancer with a Long Imaging Time Window

Abstract

Prostate cancer imaging and late-stage management can be improved with prostate-specific membrane antigen (PSMA)-targeting radiotracers. We developed a PSMA positron emission tomography (PET) radiotracer, DOTHA2-PSMA radiolabeled with 64Cu (T1/2: 12.7 h), to leverage its large imaging time window. This preclinical study aimed to evaluate the biological and imaging properties of 64Cu-DOTHA2-PSMA. Its stability was assessed in plasma ex vivo and in mice. Cellular behavior was studied for up to 48 h in LNCaP cells. Biodistribution studies were performed in balb/c mice for up to 48 h. Dynamic (1 h) and static (4 h and 24 h) PET imaging was completed in LNCaP tumor-bearing mice. 64Cu-DOTHA2-PSMA was stable ex vivo in plasma and reached cellular internalization up to 34.1 ± 4.9% injected activity (IA)/106 cells at 48 h post-injection (p.i.). Biodistribution results showed significantly lower uptake in kidneys than 68Ga-PSMA-617, our reference PET tracer (p < 0.001), but higher liver uptake at 2 h p.i. (p < 0.001). PET images showed 64Cu-DOTHA2-PSMA’s highest tumoral uptake at 4 h p.i., with a significant difference between blocked and non-blocked groups from the time of injection to 24 h p.i. The high stability and tumor uptake with a long tumor imaging time window of 64Cu-DOTHA2-PSMA potentially contribute to the prostate cancer theranostic approach and its local recurrence detection.

Keywords: DOTHA2 chelator; PET imaging; PSMA; copper-64; prostate cancer; theranostic approaches.

Scheme 1

⁶⁴Cu-DOTHA₂-PSMA synthesis.

Figure 1

⁶⁴Cu-DOTHA₂-PSMA cellular uptake and internalization (a) in comparison with ⁶⁸Ga-PSMA-617 up to 2 h p.i. and (b) without comparison, up to 48 h p.i. (c) ⁶⁴Cu-DOTHA₂-PSMA retention in comparison with ⁶⁸Ga-PSMA-617 up to 2 h p.i. and (d) up to 48 h p.i. in human adenocarcinoma cells expressing PSMA (LNCaP). ⁶⁸Ga-PSMA-617’s uptake up to 2 h p.i. was significantly lower than ⁶⁴Cu-DOTHA₂-PSMA’s (a, p < 0.001), with no significant difference in internalization and efflux.

Figure 2

(a) ⁶⁴Cu-DOTHA₂-PSMA biodistribution of non-tumor-bearing balb/c mice up to 48 h p.i. and (b) in comparison with ⁶⁸Ga-PSMA-617. Complete, numerical data are presented as Supplementary Materials. For clarity, only significant differences are shown in (b). *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 3

Representative maximal intensity projections of ⁶⁴Cu-DOTHA₂-PSMA PET in LNCaP tumor-bearing mice without (a,c,e) and with (b,d,f) the co-injection of ^natCu-DOTHA₂-PSMA as a blocking agent. (a,b) Average frames from 40 to 60 min p.i. from dynamic scans; (c,d) 4 h p.i. 20 min static scan; (e,f) 24 h p.i 1 h static scan. The 1 mm³ 3D Gaussian smoothing was applied for presentation only. Tumors are identified by circles in (b) only.

Figure 4

Time–activity curves and histograms in (a,b) tumor, in kidney (c) calyxes and (d) cortex, (e,f) liver, and (g,h) muscle for the PET imaging of NOD-Rag1^null IL2rg^null (NRG) mice and (i) biodistribution following the last scan. Note the changes in the Y-axis for better visualization. For time–activity curves and biodistributions, only significant differences are shown for clarity. ns: nonsignificant, *: p < 0.05, **: p < 0.01, ***: p < 0.001.