In this study, we conducted a preliminary investigation using PET/CT imaging to assess the performance of 64Cu conjugated with PSMA-3Q via the NOTA chelator (64Cu-NOTA-PSMA-3Q). We specifically focused on comparing the in vivo biodistribution differences between PSMA-3Q labeled with two bifunctional chelators, DOTA and NOTA.

64Cu has emerged as a highly effective PET imaging radionuclide, offering unique advantages in diagnostic imaging and therapeutic applications. Recent advancements have led to the development of several 64Cu-labeled PSMA radiotracers [19,20,21,22], which have shown significant potential for the early detection of recurrent disease and local recurrences [23]. One of the most noteworthy benefits of 64Cu is its role in the theranostic pair (64Cu/67Cu), which enables a dual diagnostic-therapeutic approach. As a diagnostic agent, 64Cu provides critical information for precision oncology, while 67Cu serves as a therapeutic radioisotope, making this pairing particularly valuable in personalized medicine. The physical properties of 64Cu further enhance its utility in PET imaging. Its extended half-life allows imaging at later time points, improving the delineation of tumors with greater accuracy and detail. This feature also facilitates the decentralized production and distribution of radiotracers, reducing logistical constraints on production sites [15]. Another unique advantage of 64Cu-labeled PSMA-targeted agents is the ability to incorporate 67Cu for therapeutic purposes within the same compound. This capability streamlines the transition from imaging to treatment, thereby supporting the development of dual-function theranostic agents. Despite the growing interest in 67Cu, its availability is currently more limited compared to the widely used 177Lu. However, efforts are underway to enhance global access to 67Cu [24]. Given its shorter half-life of 2.58 days relative to 177Lu’s 6.7 days, 67Cu is particularly suited for pharmacokinetic studies of peptide-based drugs that are rapidly excreted [25].Efficient chelation is essential for ensuring the stability and efficacy of radiotracers. Among available chelators, DOTA and NOTA are widely regarded as effective for various radionuclides due to their favorable geometry and thermodynamically stable metal-binding sites. DOTA is considered the “gold standard” chelator, capable of forming stable complexes with a broad spectrum of isotopes, such as 225Ac, 86/90Y, 177Lu, 111In, and 44/47Sc [26]. Its versatility makes it the preferred choice for chelating isotopes used in both imaging and radioligand therapy.

However, specific challenges arise when DOTA is used with 64Cu. One of the primary issues is the high liver and gallbladder uptake observed with 64Cu-labeled compounds. Research suggests that this uptake is caused by the dissociation of copper from its chelator, which is mediated by the action of superoxide dismutase (SOD) in the liver [27]. This dissociation leads to the redistribution of free copper ions, contributing to higher off-target activity. While DOTA maintains good stability with other isotopes, its in vivo kinetic stability with 64Cu is relatively lower compared to NOTA.

NOTA, on the other hand, offers a more stable chelation for 64Cu. Its superior kinetic stability minimizes copper release, leading to reduced hepatic uptake. For imaging applications where minimizing radiation dose to the liver is a priority, NOTA is the preferred chelator for 64Cu-based radiopharmaceuticals. However, for therapeutic purposes—especially when working with isotopes that require extended stability, such as 90Y or 177Lu—DOTA is more appropriate due to its ability to maintain long-term complexation [11]. The choice of chelator, therefore, depends on the specific clinical objective: if liver sparing and reduced background uptake are essential, NOTA should be used. Conversely, for radioligand therapy, where isotopes must remain bound for longer durations, DOTA remains the preferred choice.

In this study, both DOTA and NOTA were demonstrated to be effective bifunctional chelating agents (BFCAs) for the preparation of 64Cu-labeled PSMA radiopharmaceuticals used in PET/CT imaging of prostate cancer patients. However, a key distinction was observed: 64Cu-NOTA-PSMA-3Q exhibited significantly lower liver uptake compared to 64Cu-DOTA-PSMA-3Q, consistent with prior studies that have directly compared these chelators [28]. This reduction in hepatic uptake is clinically relevant, as it enhances image clarity by reducing background signal, thereby facilitating the detection of liver lesions.

The enhanced in vivo stability of 64Cu-NOTA-PSMA-3Q can be attributed to the chelation properties of NOTA. Previous studies have established that 64Cu-NODAGA-PSMA ligands exhibit greater stability than their DOTA-based counterparts, leading to improved diagnostic performance characterized by higher tumor-to-background ratios. Comparative analyses have shown that NOTA provides superior stability for 64Cu-labeled compounds compared to DOTA [29]. This increased stability is likely due to the smaller cavity size of NOTA relative to DOTA [30], which allows for a more tightly bound copper complex, thereby minimizing dissociation and off-target uptake.

From a clinical perspective, the reduced liver uptake of 64Cu-NOTA-PSMA-3Q has significant implications for prostate cancer imaging, particularly in patients with suspected liver metastases. Visceral metastases, especially in the liver, are known to be strong predictors of poor progression-free survival (PFS) and increased mortality risk [31]. As a result, the ability of 64Cu-NOTA-PSMA-3Q to provide clear, high-contrast imaging of the liver may improve the early detection and localization of metastatic lesions. This advantage could play a critical role in patient management by enabling more accurate staging and facilitating timely therapeutic intervention. Hence, 64Cu-NOTA-PSMA-3Q offers a clinically valuable diagnostic tool for the assessment of prostate cancer with potential liver involvement, supporting more precise and individualized patient care.

This study identified significant differences in the uptake of 64Cu-labeled compounds in organs with high PSMA expression, including the lacrimal, parotid, and submandibular glands. Notably, the uptake of 64Cu-labeled DOTA in these glands was substantially lower than that of 64Cu-labeled NOTA. This finding aligns with prior research by Niels J. Rupp et al. (2019), who observed that the accumulation of PSMA-targeted radiopharmaceuticals in salivary gland tissue is predominantly nonspecific [32]. Understanding the mechanisms behind this differential uptake is critical for the future design of PSMA-targeted radiopharmaceuticals, particularly in therapies using α-particle emitters with high linear energy transfer (LET).The notable difference in salivary gland uptake between 64Cu-DOTA and 64Cu-NOTA may be attributed, in part, to the pH environment of the saliva, which typically ranges from 6.5 to 7 [33]. The kinetic stability of copper chelates is known to be pH-dependent, with studies showing that Cu-NOTA exhibits greater stability than Cu-DOTA at a pH of 4.6 [11]. However, at the neutral pH found in saliva, DOTA may provide a more stable binding environment for copper, potentially resulting in reduced dissociation and uptake in the salivary glands. This pH-driven stability difference offers a plausible explanation for the observed disparity in glandular uptake between 64Cu-DOTA and 64Cu-NOTA.

From a clinical perspective, the accumulation of PSMA-targeted radiopharmaceuticals in the salivary glands has significant implications for patient well-being, particularly in the context of radionuclide therapy. Radiation-induced damage to salivary glands can result in xerostomia (dry mouth), a debilitating side effect that significantly impacts quality of life [34]. While xerostomia induced by β-emitting radionuclides like 177Lu is often reversible, irreversible gland damage has been reported with α-emitters such as 225Ac [35]. This irreversible damage poses a major challenge for dose escalation in PSMA-targeted radioligand therapy (RLT) for prostate cancer. Given these concerns, DOTA may be the preferred chelator over NOTA in therapies involving α-emitting nuclides, as its lower uptake in salivary glands could reduce the risk of radiation-induced xerostomia. For future clinical applications, particularly in intravenous radionuclide irradiation therapy, DOTA may offer a safer option when using 67Cu as the therapeutic radionuclide. By limiting uptake in non-target organs like the salivary glands, DOTA-labeled PSMA-targeted radiopharmaceuticals may enable higher therapeutic doses while mitigating adverse effects. Interestingly, the higher uptake of NOTA-labeled ligands in salivary glands may present a potential advantage for targeting tumors that originate or metastasize to these glands [36]. This dual role highlights the need for more targeted strategies to balance effective tumor targeting with reduced off-target toxicity. Further studies are required to better understand the mechanisms driving salivary gland uptake and to explore potential solutions for mitigating toxicity. These efforts are crucial for optimizing PSMA-targeted RLT protocols, particularly in patients with prostate cancer who are at risk of xerostomia due to salivary gland irradiation.

It is noteworthy that the uptake of 64Cu-NOTA-PSMA-3Q and 64Cu-DOTA-PSMA-3Q in the brain is minimal, a finding that aligns with the known properties of PSMA radioligands. This minimal uptake can be attributed to the molecular weight of PSMA, which, without the radionuclide label, ranges from 400 to 500 Da. This exceeds the typical mass transport limit of the blood-brain barrier, thereby restricting its passive diffusion into the brain. As a result, any detectable brain uptake of 64Cu-PSMA could be indicative of a disruption or compromise in blood-brain barrier integrity [23]. Despite the valuable insights provided by this study, certain limitations must be acknowledged. The most prominent limitation is the relatively small sample size, which calls for further validation using a larger cohort to confirm the generalizability of these findings. Expanding the sample size would increase the statistical power and provide a more comprehensive understanding of the biodistribution and pharmacokinetics of 64Cu-NOTA-PSMA-3Q and 64Cu-DOTA-PSMA-3Q. Another key limitation is the absence of biodistribution data from healthy volunteers. While patient-based studies are often more reflective of real-world clinical conditions, the inclusion of healthy volunteers could offer critical baseline data for comparison. Establishing a healthy biodistribution profile for these radiopharmaceuticals would provide context for interpreting patient results, particularly when assessing potential variations in uptake caused by disease-related changes, such as alterations in PSMA expression or blood-brain barrier integrity. Future research should consider the inclusion of healthy volunteer data to enhance the robustness of study conclusions and enable a more holistic assessment of the biodistribution patterns of 64Cu-labeled PSMA radiopharmaceuticals.