UCSF

While methods for early detection and risk stratification of prostate cancer (PCa) have greatly improved in recent years, there remains an unmet clinical need for improved methods to accurately detect and grade PCa non-invasively using imaging techniques. Zinc (Zn) has been studied as a target biomarker due to its unique physiology in the prostate. It accumulates in a healthy prostate to a remarkable high concentration, while it significantly decreases by 60-80% in de-differentiated PCa. Previous studies have examined this property as a potential biomarker for using imaging modalities such as MRI and fluorescence to characterize PCa, however, these techniques are not amenable to clinical translation. PET imaging has yet to be explored in detecting Zn for a tool of PCa diagnosis. This study focused on developing a novel probe, 18F-labelled Tris(2-pyridylmethyl)amine-based (18F-TPA), which exhibits excellent zinc specificity and targetability, cell membrane permeability, and low cytotoxicity. Once the cells uptake the radionuclides, they bind with intracellular free zinc ion and will not flux out of the cells. Thus, the 18F-TPA PET imaging method could directly image zinc biodistribution and therefore be applied to detect alterations in zinc homeostasis in PCa and other diseases. Methods: This project started from the chemical synthesis of a precursor compound NO2-TPA and a non-radioactive reference compound, 19F-TPA for the subsequent synthesis and characterization of radioactive 18F-TPA. 18F-TPA was synthesized in a hot cell with NO2-TPA reacting 18F in the presence of kryptofix 222 and purified using semi-prep HPLC with the conditions determined by the co-injection of NO2-TPA and 19F-TPA. The quality of 18F-TPA was ensured by co-injection with 19F-TPA to an analytical HPLC showing the same retention time. Furthermore, 19F-TPA was used to characterize Zn binding by determining a Zn19F-TPA complex formation on an analytical HPLC. Subsequently the developed probe was used to test the hypothesis of its trapping behavior when bound to Zn+2 using an in vitro cell binding assay. One group had only the probe while two other groups had TPEN, a known strong Zn chelator, and TPA that were used as blocking agents followed by the introduction of the probe. The radioactivity was measured using a Hidex gamma counter with results recording in counts per minute. Results: The 18F-TPA probe was successfully developed and purified with high yield. The cell binding assay provided evidence of the possibility the probe can be internalized when it binds to intracellular Zn+2. This was evidenced by high counts per minute (CPM) levels in cells with only probe uptake. In comparison, the cells with TPEN and TPA as blocking agents demonstrated low CPM levels. This provides preliminary evidence that the probe is Zn+2 specific. However, further in vitro assays need to be conducted with consideration of separating the membrane from the internal components of the cell. Without this consideration, it can be concluded that the probe is membrane-bound and not essentially trapped in the intracellular space of the cell. Conclusion: A new Zn+2 binding PET imaging radionuclide was developed and utilized for subsequent in vitro and in vivo assays. The in vitro cell binding assays have provided promising results that may support the hypothesis of cell internalization of the probe as it binds to Zn. The work on the in vitro assays and animal studies are still in progress. Future work will focus on additional in vitro assays and performing in vivo assays. The results from this study thus far have been encouraging on the potential of this probe as a diagnostic tool to examine zinc distribution.