| Summary: | Radiotracers that target glutamate carboxypeptidase II, also known as the prostate specific membrane antigen (GCP(II)/PSMA), have shown exceptional promise for prostate cancer imaging and molecular radiotherapy. A number of bioconjugates have been developed to target GCP(II)/PSMA which utilise the simple and robust targeting motif (Lys-C(O)-Glu) (referred to as PSMA ligand), functionalised with a chelator for radiometal incorporation. This work utilised and evaluated the tris(hydroxypyridinone) (THP) chelator due to the simplicity, speed and selectivity of its radiolabelling with gallium radioisotopes, and its potential to be developed into one-step radiopharmaceutical kits. [68Ga]Ga-THP-PSMA was assessed as a PET imaging agent and [67Ga]Ga-THP-PSMA for molecular radiotherapy due to its Auger electron emissions. The first objective was to synthesise THP-PSMA and assess its radiolabelling with gallium-68. [68Ga]Ga-THP-PSMA with over 95% radiochemical purity was produced at room temperature, and pH 7 in just 5 minutes. A one-step kit was developed, suitable for use with gallium-68 directly from a generator. [68Ga]Ga-THP-PSMA exhibited specific uptake in GCP(II)/PSMA-expressing prostate cancer cells and 50% inhibition of binding at a concentration of 361 ± 60 nM. In vivo PET imaging showed specific uptake in GCP(II)/PSMA-expressing tumours, reaching 5.6 ± 1.2 percentage injected dose per cubic centimetre. Overall, [68Ga]Ga-THP-PSMA had equivalent imaging properties but greatly simplified radiolabelling compared to other [68Ga]Ga-PSMA tracers. Clinical studies were then conducted by collaborators which found that [68Ga]Ga-THP-PSMA could delineate prostate cancer in both initial staging and biochemical recurrence. The second objective was to assess [67Ga]Ga-THP-PSMA for molecular radiotherapy. Extremely high molar activity was achieved: 326 MBq/nmol (22% chelator occupancy). Despite this, the activity delivered to GCP(II)/PSMA expressing cells was low (max 0.1 Bq/cell) and the tracer showed rapid efflux. These findings were confirmed with in vivo studies showing poor retention of [67Ga]Ga-THP-PSMA in GCP(II)/PSMA-expressing prostate cancer tumours. These results suggest that the affinity and retention, but not the molar activity, limit the use of [67Ga]Ga-THP-PSMA for molecular radiotherapy. The third objective was to determine whether replacing the urea with a thiourea in the GCP(II)/PSMA targeting motif resulted in higher affinity. This was studied using the inhibitors Glu-C(O)-Glu and Glu-C(S)-Glu, the syntheses of which are described. The in vitro inhibition assays showed that the affinity was made much weaker by the presence of the thiourea (135 μM compared to 1.34 μM) and therefore this is not an effective strategy for improving affinity. Overall the THP-PSMA bioconjugate shows excellent radiolabelling properties, with both gallium-68 and gallium-67. [68Ga]Ga-THP-PSMA is a good PET imaging agent for prostate cancer however, improved affinity for GCP(II)/PSMA and retention of the tracer may potentially enhance the utility of [67Ga]Ga-THP-PSMA for molecular radiotherapy.
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