The journey to a novel theranostic for prostate cancer : the preclinical study of 64Cu-DOTHA2-PSMA

Introduction: PSMA radiotheranostic improves metastatic and relapsing prostate cancer (PC) diagnosis and therapy. The ⁶⁸Ga/¹⁷⁷Lu-PSMA-617 pair offers promising potential but both radioligands could still be improved (e.g. through increased imaging time window or lower kidney uptake). We suggested that ⁶⁴Cu (β⁺, β⁻, Auger electrons; 12.7h half-life) could act as a theranostic radioisotope. We developed the radioligand ⁶⁴Cu-DOTHA₂-PSMA (Fig. 1A) and, based on its stability, hypothesized its efficient use for PC theranostic. Here, we look over the journey of its preclinical study, with the objectives to determine ⁶⁴Cu-DOTHA₂-PSMA’s ability to image and treat PC.

Methods: DOTHA₂-PSMA was labelled with ⁶⁴Cu(OAc)₂. Cellular assays included competition binding and cellular kinetics (uptake, internalization, efflux) in LNCaP cells. Balb/c mice were used to assess in vivo stability (n=7, ad 24h post-injection (p.i.)) and biodistribution (n=31, ad 48h p.i.). 1h dynamic PET/CT and static PET/CT (4h and 24h p.i.) were obtained with LNCaP tumor-bearing NRG mice (n=8, including 3 receiving blocking co-injection of ^natCu-DOTHA₂-PSMA). Therapy survival assays were conducted and compared to ¹⁷⁷Lu-PSMA-617 and ^natCu-DOTHA₂-PSMA (control) using the same animal model. Toxicity was assessed through weight measurement, 18 days red blood cells follow-up (comparison to normal value obtained with same protocole) and pathological examination of liver, kidneys, salivary gland and tumor (comparison to contorl and non-treated mice). Finally, dosimetry extrapolation to human was obtained based on biodistribution results using OLINDA/EXM 2.1.1.

Results: ⁶⁴Cu-DOTHA₂-PSMA was >99% radiolabeled in 5 to 10 min at room temperature with molar activities of 116±30 MBq/nmol. In vitro results showed IC₅₀ = 64±50 nM in competition to PMPA-2 and cellular uptake and internalization respectively up to 34.5±13.6 and 34.1±4.9 %IA/10⁶ cells over 48h p.i. ⁶⁴Cu-DOTHA₂-PSMA was stable in vivo up to 24h. Biodistribution showed low kidney uptake (< 15.8±7.0 %IA/g), but higher liver uptake (maximal at 30.2±4.2 %IA/g at 4h p.i.). PET demonstrated high tumoral uptake up to 24h p.i. (maximal at 23.8±11.5 %IA/cc at 4h p.i.), significantly decreased by blocking injection (p<0.05) (Fig. 1B). In therapy, survival was significantly longer using ⁶⁴Cu-DOTHA₂-PSMA (n=12) than with controls (n=10, p<0.001) (Fig. 1C). It was also longer than with ¹⁷⁷Lu-PSMA-617 (n=7), but the difference was not significant (p=0.09). One mouse treated by ¹⁷⁷Lu-PSMA-617 reached the maximal allowed weight loss. Red blood cell counts were also significantly lower than normal at days 8 and 12 p.i. with ¹⁷⁷Lu-PSMA-617 (p<0.0001), while no significant difference from normal observed with ⁶⁴Cu-DOTHA₂-PSMA. Pathological analysis showed mild, non-pathological fibrosis in kidneys, liver and salivary gland in all survival assays groups, without significant difference between groups. Small, but significant differences were noted between survival assays groups and non-treated controls (p < 0.01) (exceptions: ¹⁷⁷Lu-PSMA-617 treated group for kidneys and salivary gland). In tumor, changes such as necrosis, hemorrhage and fibrosis were present, with significantly smaller proportion of alive tumor cells in ⁶⁴Cu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617 treated mice than non-treated controls (p= 0.04 and 0.05, respectively). Finally, human estimated effective dose was 3.1x10^-2 mSv/MBq (95% CI: 2.7x10^-2– 3.6x10^-2).

Conclusions: ⁶⁴Cu-DOTHA₂-PSMA first showed high potential as a PC theranostic agent, thanks to its easy and efficient synthesis and its high in vivo stability. Results then showed that ⁶⁴Cu-DOTHA₂-PSMA offered a long imaging time window and efficiency for radionuclide therapy with acceptable toxicity and dosimetry. In the whole picture, ⁶⁴Cu-DOTHA₂-PSMA is a promising agent for PC theranostic.

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