In the past few years, theranostic radiopharmaceuticals targeting the Prostate-Specific Membrane Antigen (PSMA), which is overexpressed in the majority of prostate cancer (PCa) and its metastases, opened new avenues for the treatment and management of this disease. In particular, a variety of PSMA inhibitors were labelled with different imaging and therapeutic radionuclides, leading to the development of the β^– emitting radioconjugate ¹⁷⁷Lu-PSMA₆₁₇ (Pluvicto^â), approved by the FDA and EMA for targeted radionuclide therapy (TRT) of PSMA-positive metastatic castration-resistant PCa (mCRPC)[1,2].

More recently, ¹⁶¹Tb started to be perceived as a promising radiolanthanide and an attractive alternative to ¹⁷⁷Lu. ¹⁶¹Tb is a β^– emitter but also emits conversion electrons and Auger electrons (AE) and thus is expected to allow a combined beta and AE therapy with improved therapeutic index, particularly for disseminated cancer disease [3]. Currently, two clinical trials are underway for radionuclide therapy of prostate cancer with ¹⁶¹Tb-PSMA derivatives [4]. AEs show a high linear energy transfer (LET) over a nanometric range. Thus, the specific targeting of highly radiosensitive organelles in tumoral cells, like the nucleus or the mitochondria, might enhance the therapeutic effects of AEs at lower administered doses, while minimizing undesirable side effects [5]. Having this in mind, we have embarked on the study of dual-targeted ¹⁶¹Tb-radiocomplexes carrying a PSMA derivative and acridine orange (AO) or triphenyl phosphonium (TPP) groups to promote selective uptake by PCa cells and accumulation in the nucleus or mitochondria, respectively.

In this communication, we describe our efforts to develop ¹⁶¹Tb radiocomplexes for the targeting of the mitochondria in PCa cells, aiming to obtain improved PCa radiotheranostics with enhanced and more selective anticancer effects. We will report on dual-targeted ¹⁶¹Tb-complexes obtained with DOTA-based chelators carrying PSMA and TPP derivatives, and on their preclinical evaluation in cellular and animal tumor models.

The new radiocomplexes were obtained in high radiochemical yield and purity, and were characterized by HPLC comparison with the “cold” ^natTb congeners. Their in vitro biological evaluation included cellular uptake, internalization and PSMA-blocking studies in PSMA-positive PC3 PIP and PSMA-negative PC3 FLU cells, mitochondria uptake studies, and assessment of radiobiological effects based on the clonogenic survival and gH2AX assays. The ¹⁶¹Tb-radiocomplexes presented high PSMA-mediated cellular uptake and internalization in PCa cells. The radiobiological studies revealed that the dual-targeted complexes (¹⁶¹Tb-TPP-PSMA and ¹⁶¹Tb-TPP-G₃-PSMA) compromise cell survival in a dose-dependent manner to a higher extent than the golden-standard ¹⁶¹Tb-PSMA₆₁₇. This trend possibly reflects the highest mitochondrial uptake exhibited by the complexes containing the TPP pharmacophore.

µSPECT/CT imaging studies in mice with PSMA-positive PCa xenografts were also performed for the dual-targeted ¹⁶¹Tb-radiocomplexes and the obtained results will be discussed in terms of tumor uptake, pharmacokinetics, renal clearance and kidney retention, in order to identify the most promising ones for subsequent radiotherapeutic assays in tumor bearing mice.

In summary, the high and specific cellular uptake and internalization in prostate cancer cell lines, combined with their significant radiotoxicity, are indicative of the potential of the designed dual-targeted ¹⁶¹Tb-radiocomplexes for Auger therapy of cancer. Further studies are underway to enlighten the possible role of mitochondrial damage on their enhanced radiobiological effects.

Acknowledgements: This work was supported by Fundação para a Ciência e Tecnologia, Portugal (projects PTDC/MED-QUI/1554/2020 and UID/Multi/04349/2019) and by European Union’s Horizon 2020 research and innovation program under grant agreement 101008571 (PRISMAP). We acknowledge SCK.CEN for providing ¹⁶¹TbCl₃ and Prof. M. Pomper (Johns Hopkins Medical School, Baltimore, USA) for the gift of PC3 PIP and PC3 FLU cell lines. Joana F. Santos and Catarina D. Silva acknowledge the PhD grants PRT/BD/154612/2023 and PRT/BD/154625/2023, respectively.

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