Purpose: Hyperpolarized 13C MRI is a promising non-invasive imaging technique that can interrogate tumor microenvironments, and is amenable to clinical translation. The aim of this study was to synthesize candidate probes and evaluate their effectiveness in zinc-binding and hyperpolarized study. Methods: Candidate probes are synthesized, purified, and characterized via NMR, followed by testing in various zinc concentrations and pH buffers. Promising probes are enriched with a 13C label and evaluated with a hyperpolarizer and NMR. Results: Several probes show significant chemical shift upon binding to zinc, as well as negligible shift to different pH conditions. Conclusion: Results from this study show that molecular imaging of zinc is possible, but require further optimization of 13C compound synthesis routes and hyperpolarized experiments.

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.

Background: The acidification of the tumor microenvironment is a result of extensive

metabolic reprogramming in cancer cells and is linked with tumor metastasis. Hyperpolarized

MRI is a method for imaging and quantifying this change in pH, but suffers from rapid signal

loss from spin-lattice (T1) relaxation. We propose using hydrogen/deuterium exchange on

hyperpolarized 13C probes in order to prolong hyperpolarized signal by reducing T1 relaxation.

Methods: H/D exchange was performed on several amino acids and amino acid derivatives

with utility in HP-MRI. Isotopic enrichment was evaluated using 1H NMR. The T1 relaxation

constant was quantified by analyzing the decay of hyperpolarized signal of deuterated vs nondeuterated 13C compounds. Results: H/D exchange was successfully used to enrich compounds

with deuterium with high isotopic enrichment and moderate to high chemical yield. The T1

relaxation constant of all fully analyzed 13C compounds exhibited a significant increase after

deuteration at 3T: T1 of 13C Gly increased from 52.0±3.2 to 65.0±1.2s, 13C Ala from 52.9±2.2

to 66.4±1.7s, 13C Val from 38.1±1.1 to 49.2±0.4s. Conclusion: H/D exchange method

described is a viable technique for inexpensive and direct deuterium labeling. Deuterium

labeling be applied to hyperpolarized 13C MRI probes to prolong HP signal by lengthening T1.

Current treatments for prostate cancer, such as chemotherapy and androgen receptor signaling inhibitors, have varying success rates with unpredictable outcomes. As a result, there is a need for the development of novel targeted therapies which can provide positive responses from patients. Radioligand therapy (RLT) in combination with poly (ADP-ribose) polymerase (PARP) inhibitors has shown efficacy in preliminary in vitro studies. In this study, we will use RLT with beta emitter 177-Lu and alpha emitters 227-Th and 225-Ac conjugated to a CD46 targeting antibody, in addition to Niraparib or Talazoparib as the PARP inhibitor. MTT and colony forming assays were performed on two prostate cancer cell lines. 225-Ac-YS5 in combination with Talazoparib showed a promising ZIP synergy score of 25.35, indicating that the two work synergistically to provide better therapeutic outcomes. After in vitro efficacy, we will test in murine models, with a future goal of a human clinical trial.

Prostate-specific membrane antigen (PSMA) is a cell surface enzyme highly over expressed in prostate cancer cells that can be employed as a target for prostate cancer imaging and drug delivery. Boron Neutron Capture Therapy (BNCT) is an emerging noninvasive therapeutic modality for treating locally invasive malignant tumors by selective delivery of high boron content to the tumour and then subjecting the tumour to epithermal neutron beam radiation. In this study, we develop carborane encapsulated amphiphilic polymer nanoparticles by conjugating urea based PSMA inhibitors (ACUPA) and 89Zr chelating deferoxamine B (DFB) ligand and have investigated their efficacy to deliver enhanced boron payload to PSMA positive prostate cancer cells with simultaneous positron emission tomography (PET) imaging . Three different carborane encapsulated PLGA-b-PEG nanoparticles (NPs) were formulated with and without the PSMA targeting ligand, out of which two selected formulations; DFB(25)ACUPA(75) NPs and DFB(25) NPs radiolabelled with 89Zr were administered to mice bearing dual PSMA(+) PC3-Pip and PSMA(-) PC3-Flu xenografts. PET imaging and biodistribution studies were performed to demonstrate the in vivo uptake in mice. The NPs showed 2-fold higher uptake in PSMA(+) PC3-Pip tumors to that of PSMA(-) PC3-Flu tumors with a very high tumor/blood ratio of 20. However, no significant influence of the ACUPA ligands were observed. Additionally, the NPs demonstrated fast release of carborane with low delivery of boron to tumors in vivo. Although the in vivo afficacy of those NPs remain limited, a significant progress towards the synthesis, characterization and initial biological evaluation of the polymer nanoparticles is proposed in this report and the results presented could guide the future design of amphiphilic polymer NPs for theranostic applications.

Multiple myeloma is one of the most common blood cancers. Many drugs have been developed to treat multiple myeloma, yet refractory and relapsed disease remain prevalent. The recent identification of CD46, an antigen overexpressed on multiple myeloma cells, has led to new development of antibody-based immunotherapies, including the antibody-drug conjugate CD46 ADC and the radioimmunotherapy [225Ac]Ac-Macropa-PEG4-YS5. The structures of both drugs include the CD46-binding monoclonal antibody YS5, and both have shown high tumor binding and antitumor efficacy in pre-clinical models for multiple myeloma. A Phase 1 clinical trial is also underway to evaluate CD46 ADC in multiple myeloma. While initial results are promising, both drugs have noted drawbacks, with blood cancers like multiple myeloma known to develop resistance to ADCs over time and [225Ac]Ac-Macropa-PEG4-YS5 studies displaying nephrotoxicity at higher doses. Hypothesizing that the use of the two drugs in tandem could improve therapeutic efficacy, this study investigated the performance of RIT & ADC combined treatment across preclinical multiple myeloma models in-vitro and in-vivo. Results of initial cell-based assays demonstrated a high cell killing ability of combination treatment, with evidence of a synergistic interaction between drugs at select combination concentrations. A pilot xenograft mouse model comparing combination treatment to RIT & ADC monotherapies showed a widespread reduction in tumor burden with animal body weight remaining stable, potentially indicating reduced off-target toxicity. Future work will monitor the antitumor efficacy of RIT & ADC combination therapy over a longer time scale and measure organ damage ex-vivo to further compare against prior monotherapy results. This encouraging initial data provides guidance on combination dosing regimens for future preclinical research and suggests that RIT & ADC combination therapy may be a valuable clinical option for treating multiple myeloma.