Synthesis of novel PSMA ligands and preclinical evaluation of [99mTc]TcO-ABX474, a radioligand for SPECT imaging of prostate cancer

Objectives: During the last 15 years several PSMA PET ligands for prostate cancer imaging have been developed resulting in the recent approvals of 68Ga-PSMA-11, 18F-PSMA-1007 and 18F-DCFPyL.[1,2] However, 99mTc remains a popular radiometal for SPECT imaging due to its longer half-life and availability from 99Mo/99mTc generators especially in outpatient imaging centers. Due to the widespread availability of SPECT cameras PSMA SPECT ligands have the potential to substantially extend the availability of PSMA-based imaging, but currently no PSMA SPECT ligand is approved. MIP-1404, PSMA-I&S and HYNIC-iPSMA are rare examples of PSMA SPECT tracers in clinical development. Accordingly, the aim of our study was to develop a PSMA ligand for 99mTc SPECT imaging of prostate cancer based on an N2S2 chelator, which enables reliable radiolabeling to form stable 99mTc complexes.[3]

Methods: A number of compounds, which contain the PSMA binding motif Glu-urea-Lys, a varying linker and a bisaminothiol (BAT)-type N2S2 chelator, were synthesized. Selected compounds were radiolabeled with 99mTc, followed by assessment of in vitro stability of the formed complexes via radio-HPLC. The specific binding affinity towards PSMA and the internalization of the radioligands were examined in LNCaP cells, supplemented by autoradiographic studies. Tissue distribution and tumor accumulation were evaluated in LNCaP-tumor bearing mice via quantitative SPECT/CT imaging, benchmarked with [99mTc]Tc-PSMA-I&S[4] and [68Ga]Ga-PSMA-11.

Results: Among the six 99mTc radioligands synthesized and examined, [99mTc]TcO-ABX474 showed most favorable properties. Radiolabeling of ABX474 (50 µg) was achieved starting from [99mTc]NaTcO4 (0.2-2 GBq) in saline (1.4 mL) using SnCl2 (1 µg in 0.01 M HCl) as reducing agent, in presence of calcium-D-heptagluconate (10 µg) and D-mannitol (1 mg) at pH 5-6. Incubation at r.t. for 20 min followed by heating at 80°C for 20 min afforded the product-containing solution with a radiochemical purity of 93.8 ± 3.1 % (n=20) optimization planned during kit development). [99mTc]TcO-ABX474 was stable in DPBS (96.7 ± 2.1 % unchanged, n=3) at 25°C for 20 h as well as in mouse plasma (96.7 ± 1.3 % unchanged, n=3) and human plasma (91.7 ± 0.3 % unchanged, n=5) at 37°C for 6 h, respectively. [99mTc]TcO-ABX474 showed high binding affinity towards PSMA (Kd= 7.2 ± 1.7 nM) and substantial uptake in LNCaP cells (binding: 3.2 %AD/mg protein internalization: 2.8 %AD/mg protein, 47% internalization of total cell bound activity at 37°C).
In LNCaP xenograft mice, [99mTc]TcO-ABX474 showed high PSMA-specific tumor uptake, mainly renal excretion and moderate kidney retention. Hence, [99mTc]TcO-ABX474 exhibited higher tumor-to-background ratios (SUV) between 1‒4 hours after injection (tumor/muscle: 25.2‒52.7 tumor/kidney: 0.3‒0.37) compared to the reference compound [99mTc]Tc-PSMA-I&S (tumor/muscle: 5.7‒18.2 tumor/kidney: 0.17‒0.2). [99mTc]TcO-ABX474 allowed for similar tumor visualization compared to PET/CT imaging with [68Ga]Ga-PSMA-11 (tumor/muscle: 22.9 tumor/kidney: 0.24) 1 hour after injection.

Conclusion: This study demonstrates that [99mTc]TcO-ABX474 is a promising radiotracer candidate for PSMA-specific SPECT imaging of prostate cancer warranting further clinical evaluation.

Acknowledgments: The authors would like to thank the Sächsische Aufbaubank - Förderbank - for financial support (100363946).

References:

[1] https://www.fda.gov/drugs/news-events-human-drugs/fda-approves-second-psma-targeted-pet-imaging-drug-men-prostate-cancer, published on 27/05/2021.

[2] https://www.has-sante.fr/jcms/p_3337433/en/radelumin-18f-psma-1007-cancer-de-la-prostate, published on 04/05/2022.

[3] Hoepping, A. et al., EPO Patent Application EP22174909.6, 23/05/2022.

[4] Robu, S. et al., J Nucl Med 2017, 58, 235-242.