Summary

Radiolabeled prostate-specific membrane antigen (PSMA)-targeting agents hold promise for prostate cancer diagnosis and therapy. However, their tendency to accumulate in salivary glands poses challenges due to dose-limiting xerostomia. JB-1498, with its highly negatively charged linker, offers a potential solution. This study aims to compare the salivary gland uptake of [68Ga]Ga-JB-1498 to [68Ga]Ga-PSMA-11 in murine models, shedding light on its pharmacokinetic behavior and potential as a safer therapeutic option for prostate cancer.

Prostate cancer stands as one of the most prevalent cancers among men, posing significant mortality rates globally. Metastatic castration-resistant prostate cancer, in particular, presents a grim prognosis, with a substantial number of deaths attributed to it annually. Glutamate carboxypeptidase II (GCP-II), also known as prostate-specific membrane antigen (PSMA), emerges as a key target due to its high expression in prostate cancers.

Advancements in positron emission tomography (PET) imaging utilizing PSMA-targeting tracers have revolutionized the non-invasive detection of metastatic prostate cancer. Moreover, therapeutic trials employing PSMA-targeting agents have shown promising results, notably demonstrated in the VISION trial utilizing [177Lu]Lu-PSMA-617, leading to FDA approval. However, despite its efficacy, current therapeutic regimens such as [177Lu]Lu-PSMA-617 still pose challenges, including dose-limiting xerostomia resulting from salivary gland radiation exposure.

Efforts to mitigate salivary gland toxicity include exploring alternative therapeutic agents like [225Ac]Ac-PSMA-617 and [177Lu]Lu-PSMA-I&T, alongside various strategies aimed at reducing salivary gland uptake of PSMA-targeting agents. These strategies range from external cooling to the administration of PSMA inhibitors and other pharmacological interventions. However, emerging evidence suggests that modifying the molecular structure of PSMA-targeting ligands may offer a promising avenue to decrease salivary gland uptake.

In this study, they present preliminary data from murine models, exploring the potential of constructing PSMA-targeting molecules with highly negatively charged linkers as a novel strategy to reduce salivary gland uptake, thus addressing a critical limitation in current therapeutic approaches for prostate cancer.

Materials and Methods

Chemicals used in this study were of reagent grade quality, as specified in Additional file 1. JB-1498 (see Fig. 1) was synthesized based on the structure "5" reported by Huang et al., with modifications involving the substitution of the Bn-NOTA chelate with DOTA (Huang et al., 2014). The synthesis of JB-1498 employed Fmoc-based solid-phase peptide synthesis, utilizing Rink amid resin as the solid support. Purification of JB-1498 was carried out via high-performance liquid chromatography (HPLC), following detailed procedures outlined in Additional file 1.

Structure of JB-1498, a PSMA/GCP-II targeting molecule with a highly negatively charged linker between the urea and the DOTA chelate

The binding affinity of JB-1498 to GCP-II was assessed using a fluorescent enzymatic inhibition assay, following the methodology outlined by Kozikowski et al. (2004), with detailed procedures provided in Additional file 1.

Radiolabeling of JB-1498 was achieved by heating it in aqueous sodium acetate solution at pH 4–5, typically at temperatures exceeding 95°C for 7–10 minutes. The reaction conditions were optimized iteratively. For the initial biodistribution study, JB-1498 was labeled with [68Ga]GaCl3 obtained from a spent 68Ge/68Ga generator, whereas a fresh Gallium-68 generator was utilized for the high molar activity study.

In the biodistribution studies, mice were injected with the radiolabeled JB-1498 solution and euthanized at specified time points. Major organs were dissected for radioactivity quantification. Tumor-bearing and non-tumor-bearing mice were utilized to evaluate JB-1498 uptake under different conditions.

Results from nanoScan® PET/CT

For micro-PET imaging, high molar activity [68Ga]Ga-JB-1498 and [68Ga]Ga-PSMA-11 were administered to non-tumor-bearing mice via tail vein injection. Imaging was conducted using a nanoScan PET/CT instrument (Mediso Ltd.), with PET acquisition performed for 45 minutes starting 25 minutes post-injection.

Detailed procedures for radiolabeling, biodistribution studies, and imaging are provided in Additional file 1, along with figures illustrating HPLC traces and experimental setups.

In the biodistribution studies, JB-1498 exhibited favorable tumor uptake while demonstrating significantly reduced accumulation in the salivary glands and liver. Specifically, the initial study utilizing an aged ITG Ga-68 generator showed minimal salivary gland uptake (0.13 ± 0.01%) compared to tumor uptake (12.09 ± 1.28%). Subsequent biodistribution studies with high molar activity [68Ga]Ga-JB1498 reaffirmed this trend, with salivary gland uptake remaining notably low at 0.39 ± 0.24% ID/g, while kidney activity was observed at 10.12 ± 1.73% ID/g.

Comparatively, the uptake values of JB-1498 in salivary glands and kidneys were markedly lower than those reported for [68Ga]Ga-PSMA-11 in the literature. Micro-PET images acquired one hour post-injection of [68Ga]Ga-JB-1498 and [68Ga]Ga-PSMA-11 visually confirmed this distinction, showcasing notably reduced accumulation in both salivary glands and kidneys with JB-1498 compared to PSMA-11.

These findings highlight the potential of JB-1498 as a promising PSMA-targeting agent with reduced salivary gland and kidney uptake, thereby holding implications for enhancing the safety and efficacy of radiolabeled PSMA-targeted therapies.

Left panel: [68Ga]Ga-PSMA-11 and [68Ga]Ga-JB-1498 head-to-head comparison micro-PET images at 1 h post injection, with same SUV grayscale. There is very little salivary gland (SG) and background activity in the mouse injected with [68Ga]Ga-JB-1498 compared to [68Ga]Ga-PSMA-11. Most of the injected [68Ga]Ga-JB-1498 is in the urinary bladder due to rapid tracer excretion via the kidneys. Right Panel: representative time-activity curve of [68Ga]Ga-PSMA-11 and [68Ga]Ga-JB-1498 in salivary glands (combined parotid and submandibular glands) and kidneys during the first hour of micro-PET imaging. The diverging time activity curve reflects active salivary gland and renal cortical uptake of [68Ga]Ga-PSMA-11 and lack of specific uptake of [68Ga]Ga-JB-1498 in these two organs

Reducing salivary gland toxicity associated with current GCP-II targeted treatments has been a longstanding challenge. Experimental systems for testing a compound's propensity for salivary gland uptake have only recently become available, with studies by Rousseau et al. (2018) and Roy et al. (2020) providing valuable insights using mouse models.

The study builds upon prior work, where GCP-II inhibitors with highly negatively charged linkers were designed, initially demonstrating promising results in reducing organ activity. The subsequent construction of JB-1498, a variant of the molecule with enhanced properties, further validated this approach. The initial biodistribution studies with [68Ga]Ga-JB-1498 showed minimal salivary gland and renal cortical uptake, consistent with previous findings and in contrast to [68Ga]Ga-PSMA-11.

To explore the impact of molar activity on tracer uptake, additional biodistribution studies were conducted using high molar activity [68Ga]Ga-JB-1498, as specified in previous literature. Despite a slight increase in salivary gland uptake compared to lower molar activity experiments, [68Ga]Ga-JB-1498 still exhibited significantly lower uptake than [68Ga]Ga-PSMA-11. Dynamic micro-PET imaging further highlighted these differences in pharmacodynamic behavior.

The mechanisms underlying salivary gland uptake of PSMA-targeting ligands remain unclear, and the findings suggest that structural modifications may offer promising avenues for reducing uptake. Future studies exploring different regions of the molecule, such as the glutamate sidechain and overall charge in the linker region, could provide further insights.

While the findings in mice are promising, caution is warranted regarding translation to human settings. Nevertheless, the potential for structural modifications to mitigate salivary gland toxicity and expand the therapeutic window of PSMA-targeted therapies is encouraging, highlighting avenues for future investigation.

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