Nuclear Medicine Based on the Example of [89Zr]Zr-PSMA-DFO: Comparison
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Please note this is a comparison between Version 2 by Mona Zou and Version 1 by Dr. Klaus Schomäcker.

The interdisciplinary possibilities inherent in nuclear medicine offer an opportunity for the patient-centered development of radioactive pharmaceuticals based on specific research questions. This approach provides radiopharmaceutical manufacturers with a robust scientific foundation on which to navigate the regulatory requirements for drug approval laid down by the law. A vivid illustration of this interdisciplinary cooperation has been the development of a Zr-89-labeled PSMA ligand where reliable results have been obtained across various domains, including chemistry, radiochemistry, biochemistry, and preclinical research. This comprehensive process extended to feasibility studies conducted with carefully selected patients from a single nuclear medicine clinic. 

  • translational nuclear medicine
  • prostate carcinoma
  • PSMA
  • zirconium-89
  • PET

1. Insights into the World of Radiopharmacy

The development of radiopharmaceuticals is a complex, multistage process. Some of these stages are subject to strict regulatory requirements and monitoring measures.
The process typically begins with the identification of a medical need. The role of nuclear medicine at this stage is to provide a means of early disease detection and targeted therapy.
The selection of an appropriate radionuclide that will achieve the desired imaging or therapeutic outcome is crucial here. Factors such as the physical properties, half-life, emission characteristics, and decay mode need to be carefully considered and the availability and cost of the radionuclide assessed to ensure practicality and feasibility. The feasibility of radiolabeling the chosen radionuclide is also a factor. Designing a nonradioactive precursor that will bind stably to the radionuclide is crucial. Quality control establishes the identity of radiopharmaceutical products and guarantees their purity and the safety when using them, while optimization of the radiolabeling methods maximizes reproducibility.
Biochemical and cell biological studies evaluate the affinity and specificity of candidate radiopharmaceuticals for target cells, while preclinical studies provide information on their biodistribution, pharmacokinetics, and efficacy.
Radiation physics is used to determine the best radiation dose for patients and to optimize the delivery of radiation to the target site, while a pharmacological approach is needed to transfer production to specific devices and to set up quality management and control procedures.
Clinical studies investigate the safety, efficacy, and clinical endpoints of the use of radiopharmaceuticals under consideration. Ethics approval, patient selection, safety monitoring, and data analysis are all part of this process.
Furthermore, each step of radiopharmaceutical development is subject to specific regulatory requirements to ensure a safe and effective development of radiopharmaceuticals up to the stage of marketing authorization.

2. What Innovative Nuclear Medicine Concept Lies behind the Development of [89Zr]Zr-PSMA-DFO?

In many cases of prostate cancer, the levels of prostate-specific antigen (PSA) rise again after patients have undergone surgery or radiation therapy, a phenomenon known as biochemical recurrence (BCR). Early detection of these recurring tumor lesions is crucial in order to make the right treatment decisions and improve the patient’s chances of survival.
Positron emission tomography/computed tomography (PET/CT), using radioactively labeled ligands specific to the prostate-specific membrane antigen (PSMA) is commonly employed to locate prostate cancer after BCR. This imaging procedure achieves more reliable detection rates than other imaging techniques. However, it is worth noting that PSMA PET scans fail to localize tumor lesions in approximately 20% of the patients with BCR [1]. The PSA level can also influence the likelihood of a positive PSMA-PET. A meta-analysis of 37 studies found that the detection rate of PSMA-PET in patients with a PSA level of less than 0.5 ng/mL was 36% but reached 86% in patients with a PSA level of more than 2 ng/mL. [2].
The reason for this is simple: Established PSMA tracers based on Ga-68 or F-18 are of limited use due to their short radioactive half-life, which requires PET scans to be performed within 3 h of injection. In this short time period, it is seldom possible to achieve the necessary contrasts needed for imaging, as more time is required for adequate tumor accumulation and effective clearance from the blood and surrounding tissues. Imaging based on nuclear medicine can be achieved even when the PSMA expression is comparatively low, as is the case in biochemical recurrence.
To address this, the researchers developed a new PSMA tracer called [89Zr]Zr-PSMA-DFO, which has a much longer radionuclide half-life (77 h), allowing imaging to be carried out several days after injection when the background activity is minimized [3,4][3][4].

3. (Radio)chemistry

Radiochemical processes facilitate the binding of the radioactive Zr-89 radionuclide to the PSMA molecule, enabling doctors to track and visualize prostate cancer in the body using positron emission tomography (PET) scans.

3.1. Radionuclide Selection: What Is Special about Zr-89?

The radionuclide Zr-89-zirconium, with a half-life of 3.2 days, presents a valuable improvement to BCR PET imaging, particularly in combination with PET tracers labeled with short-lived isotopes like [68Ga]-PSMA11- or F-18-labeled PSMA ligands. Traditionally, Zr-89 has been primarily employed for labeling antibodies [5] and less so for small peptide molecules targeting PSMA (prostate-specific membrane antigen). Our innovation centers around the development of the [89Zr]Zr-PSMA-DFO construct for delayed PSMA-PET imaging (48 h postinjection).
This delayed imaging approach allows small tumor lesions or lesions with limited PSMA expression to be identified, as these may require more time for the PSMA inhibitor to internalize. Furthermore, it provides a means of detecting tumors that was previously overlooked due to their proximity to the ureter, following renal clearance and the associated background activity in the urine.
Zr-89 predominantly decays through positron emission (23%) and electron capture (77%) to yield the stable isotope Y-89. The emitted positrons exhibit a maximum energy of 897 keV and an average energy of 396.9 keV, which is sufficiently low to generate PET images with high spatial resolution (transverse and axial spatial resolutions at 1 cm are 4.5 and 4.7 mm for Zr-89, respectively). The potentially disruptive effects of spontaneous gamma emissions from Zr-89, emitting photons at 908.97 keV (in 99% of cases), can be counteracted by adjusting the energy window of the PET scanner [6,7,8,9][6][7][8][9].

3.2. The Nonradioactive Precursor: Why Specifically PSMA-DFO?

In our pursuit of a PSMA inhibitor compatible with zirconium-89, the researchers have identified a precursor known as PSMA-DFO (EuK-2Nal-AMCH-N-sucDFO-Fe). Manufactured by ABX Radeberg in close proximity to Dresden, this compound represents an alternative to the PSMA-617 precursor initially designed for labeling with the beta-emitter Lu-177. The sole distinction between the two lies in the replacement of the DOTA chelator with Desferrioxamine (DFO). The suitability of this molecule for coupling Zr-89 to antibodies has been convincingly demonstrated [8].
However, the incorporation of zirconium-89 into the nonradioactive PSMA-617 precursor modified with DFO presented a challenge due to the presence of an iron atom at the designated site intended for the Zr-89 introduction. The iron must therefore be removed before radioactive labeling is performed. This is imperative. Conversely, commercially available [89Zr]Zr-oxalate can be used to label this precursor efficiently.
Reports from various research groups now describe the direct labeling of PSMA-617 with zirconium-89. This involves the chlorination of [89Zr]Zr-oxalate [10,11][10][11]. Some experience of its use in a clinical setting has also been documented [12,13][12][13].

4. Radiotracer Development/Production

4.1. Radiolabeling/Quality Control/Stability

Details of these features are given by our group elsewhere [3]. Figure 1 provides an overview of the chemical processes involved in the preparation and execution of the DFO-modified PSMA-617 analog.
Molecules 29 00185 g001
Figure 1. Process flow for labeling of EuK-2Nal-AMCH-N-sucDFO-Fe with Zr-89.
For the radiolabeling step, the pH of a solution containing Zr-89 is adjusted using specific chemicals. Unbound Zr-89 is then removed efficiently by solid-phase extraction. To test the stability of the PSMA-targeting radiotracer, 100 μL of [89Zr]Zr-PSMA DFO are mixed with 1 mL of either PBS or human serum. These mixtures are kept at a constant temperature of 37 °C by means of a heating block for different time periods. Radio thin-layer chromatography is used to check the radiochemical purity of the samples at various intervals: immediately after reaching 37 °C and at 1 h, 2 h, 24 h, 48 h, and 72 h later up to 7 days. The complexes of interest stay in place on the chromatographic strip, while unbound Zr-89 moves with the mobile phase.
The labeling of [89Zr]Zr PSMA DFO is performed to a radiochemical purity of ≥99.9%. It shows very high stability in phosphate-buffered saline (PBS) and human serum, lasting for up to 7 days at 37 °C.

4.2. Optimization

The multistage production of [89Zr]Zr-PSMA-DFO is relatively complex (Figure 1). One particularly time-consuming aspect of the labeling process is the removal of iron from the precursor Fe-N-sucDFO-PSMA. In addition to the time factor here, there is also the question of how well this multistage process adheres to Good Manufacturing Practice (GMP) guidelines. Ideally, the actual coupling of Zr-89 to the PSMA ligand should take place using a synthesis module.
Such synthesis modules have been specifically designed to enable the versatile and efficient routine production of various radiopharmaceuticals while adhering to GMP requirements. The concept of disposable cassettes pursued in this approach allows the user to label different radionuclides on the same synthesis device without the risk of cross-contamination. The disposable cassettes include all necessary vessels, reagents, and purification columns. The automated process concludes with a final sterile filtration, including a filter integrity test. Iron removal should therefore take place before the actual labeling process. The resarchers wanted to verify whether it is possible to establish a stock of an iron-free PSMA-DFO precursor, stored in an aqueous solution at 4 °C in the refrigerator, and to determine its shelf life. This was done by investigating whether the iron-free precursor can be labeled with zirconium-89 after several months of storage without compromising the yield, cell binding, and internalization.
It can be demonstrated that the chemical reactivity, specific binding, and internalization using the iron-free precursor remained unaffected after storage at 5 °C in an aqueous solution for a period of 6 months. This result demonstrates the feasibility of creating a stock solution of N-sucDFO-PSMA. Consequently, [89Zr]Zr-PSMA-DFO can be efficiently and reliably produced in accordance with GMP guidelines in a single step using an automated synthesis module.

5. Biochemistry/Cell Biology

The investigations primarily focused on affinity, cell binding, and internalization studies, the objective being to demonstrate that the Zr-89-PSMA ligand was at least as effective with regard to these properties as the established PSMA ligands labeled with the short-lived radionuclides F-18 and Ga-68.

5.1. Affinity and Binding Assay

The affinity was determined by measuring the equilibrium dissociation constant, KD. This is a measure of how strongly a ligand binds to its target. It quantifies the balance between the association (binding) and dissociation (release) of the ligand from its target molecule. A lower KD value signifies a stronger binding affinity, indicating that the ligand interacts in a more stable manner with its target. In essence, KD provides crucial information about the strength of the molecular interaction between the ligand and its target.

5.2. Specificity of Cell Binding and Internalization

High specificity is synonymous with the targeted detection of prostate tumor lesions and enhances the diagnostic accuracy by avoiding false positives or false negatives. Additionally, it enables precise, focused therapy when using beta-emitting PSMA ligands, sparing healthy tissue. Specificity minimizes undesirable side effects and toxicities, thereby improving treatment tolerability. Lastly, it helps to avoid unnecessary examinations and interventions, ultimately increasing the efficiency and safety of prostate cancer diagnosis and therapy with radioactive PSMA ligands, thereby benefiting patients. Internalization has been a crucial parameter in assessing [89Zr]Zr-PSMA-DFO in relation to other PSMA ligands, as it indicates how effectively the ligand enters and interacts with the cells, which has a direct impact on its performance for diagnostic and therapeutic purposes. It also helps to determine the ligand’s ability to target and potentially treat cancer cells.
To gain insights into the specificity of our Zr-89-labeled PSMA ligand, the resaerchers conducted experiments to assess its effectiveness in recognizing its specific target, PSMA (prostate-specific membrane antigen), and in entering and binding to prostate cancer cells. Our objective was to compare this performance with well-established PSMA ligands currently utilized in clinical practice.
In these experiments, the resaerchers observed that cells bind to our ligand in both specific and nonspecific ways. To understand how much of this binding is specific and how much is not, the resaerchers used a control substance called 2-PMPA. 2-PMPA, short for 2-(phosphonomethyl)pentanedioic acid, is a well-known inhibitor of PSMA that is frequently used in scientific studies and experiments to block PSMA activity. This is put to use when testing the specific binding of PSMA ligands and ensuring that the measured effects are indeed a result of the interaction with PSMA.

5.3. Interim Summary

In all the in vitro parameters examined, including affinity (KD values), specific cellular binding, and internalization, the results for the newly developed Zr-89 ligand closely mirrored those obtained with established radioactive PSMA ligands. The exception here was [99mTc]Tc-PSMA I&S, especially in terms of the KD value.
At this point on our journey “from bench to bedside”, the resaerchers found ourselves at a critical juncture, as the resaerchers needed to decide whether to continue the comparison of Zr-89 with the established reference tracers in a living organism, specifically in animal experiments. To minimize the number of experimental animals used, the resaerchers decided to carry out further comparisons with the F-18- and Ga-68-labeled PSMA ligands alone.

6. Preclinical Research

6.1. Animal Experiments: Tumor/Background Ratios

An overarching aspect of animal experiments is the evaluation of the tolerance to and potential side effects of the candidate radiopharmaceutical under investigation.
In the animal-based segment of the study, our specific aim was to test the hypothesis that [89Zr]Zr-PSMA DFO could achieve higher tumor-to-background ratios than the short-lived tracers under investigation.
The term “tumor-to-background ratio” refers to the ratio of radioactive signal intensity or radioactivity concentration within the tumor (the target region) compared to the surrounding area, often referred to as the “background”.
A higher tumor-to-background ratio means that the radioactive signal within the tumor is more pronounced and distinct than in the surrounding normal or background tissues. This is crucial for the imaging quality, as it enhances the ability to detect and locate the tumor accurately. Where the tumor-to-background ratio is low, identifying the tumor can become a challenge, rendering imaging less reliable.
The aim of our investigation was to determine whether the Zr-89-labeled PSMA ligand can generate a higher tumor-to-background ratio than other tracers. The higher this ratio is, the more precise and reliable the imaging will be, and this is of paramount importance for the diagnosis and monitoring of cancer.
Details of how these investigations were conducted and the results obtained have been published extensively elsewhere [3]. The resaerchers will therefore provide just a brief summary of the experimental procedures used and highlight the key findings. Among the animal-based studies, both biodistribution in animal tissue and small animal PET scans in mice bearing a LNCaP tumor xenograft were conducted.
Biodistribution studies assessed radioactivity accumulation in various tissues and calculated the tumor-to-blood, tumor-to-muscle, and tumor-to-kidney ratios. In adherence to EU directive 2010/63/EU and regional authorities’ approval, only 35 male CB17-SCID mice (age: 6 weeks, weight: 17–20 g) were used. Tumor cells were implanted subcutaneously into the right flank, and once the tumors had reached 300 to 600 mg, the mice were sacrificed for biodistribution studies or PET imaging.
On the experiment day, each mouse received intravenous injections of [89Zr]Zr-PSMA-DFO, [18F]F-JK-PSMA-7, or [68Ga]Ga-PSMA-11. The mice were sacrificed at specified time points, and their organs (blood, liver, spleen, kidneys, muscle, bone, thyroid, lungs, intestines, tumor, heart, and prostate) were dissected and weighed and radioactivity measured. The biodistribution studies were conducted at 2, 4, and 24 h after the injection of [89Zr]Zr-PSMA-DFO, [18F]F-JK-PSMA-7, or [68Ga]Ga-PSMA-11. These time intervals were chosen to accommodate the short physical half-lives of Ga-68 and F-18 while simultaneously comparing the performance of the Zr-89-labeled PSMA ligand with those labeled with short-lived radionuclides. Furthermore, these intervals highlight the superiority of the Zr-89-labeled tracer after an extended administration period (24 h). The results are expressed as a percentage of the injected dose per gram of tissue (% ID/g) (n = 5). After 24 h, [89Zr]Zr-PSMA-DFO displayed much higher ratios between the tumor and the surrounding areas (tumor/blood: 309 ± 89, tumor/muscle: 450 ± 38) in LNCaP tumor xenografts when compared to [68Ga]Ga-PSMA-11 (tumor/blood: 112 ± 57, tumor/muscle: 58 ± 36) or [18F]F-JK-PSMA-7 (tumor/blood: 175 ± 30, tumor/muscle: 114 ± 14) after only 4 h (p < 0.01).

6.2. Animal Experiments: Small Animal PET

After the biodistribution experiments, the resaerchers used PET scans to confirm the biodistribution findings. For this purpose, PET scans were obtained of LNCaP tumors in mice with [68Ga]Ga-PSMA-11 and [18F]F-JK-PSMA-7 tracers, each mouse receiving 10 MBq of the tracer.
The resaerchers also checked to see whether [89Zr]Zr-PSMA-DFO specifically binds to PSMA by using a PSMA blocker called 2-PMPA. Some mice received 2-PMPA, while some did not. All scans were done while the mice were under anesthesia.
The scans lasted for 60 min and began 60 min after the tracer injection. Further scans were carried out at 4 h, 21 h, and 48 h after the injection of [89Zr]Zr-PSMA-DFO. These scans helped us to see where the tracer went in the body. The summed images were reconstructed using an iterative OSEM3D/MAP procedure resulting in voxel sizes of 0.47 × 0.47 × 0.80 mm. Postprocessing and image analyses were performed with VINCI 4.72 (Max-Planck-Institute for Metabolism Research, Cologne, Germany). The images were Gauss-filtered (1 mm FWHM) and intensity-normalized to the injected dose, corrected for body weight (SUVbw). Finally, the resaerchers adjusted the images based on the amount of tracer used in relation to the weight of the mouse and thereby achieved better results.
In live, small animal PET imaging, the resaerchers observed that the ability to visualize tumors with [89Zr]Zr-PSMA-DFO was similar to that achieved with [68Ga]Ga-PSMA-11 or [18F]F-JK-PSMA-7 at early time points (1 h after injection). Furthermore, PET scans performed up to 48 h after injection continued to provide clear tumor visualization, even at later time points [3].

6.3. Toxicity

To rule out acute toxicity of the nonradioactive precursor, the resaerchers commissioned a certified external laboratory to test this. A group of 10 mice were injected intravenously with 1 mg/kg body weight of the nonradioactive precursor EuK-2Nal-Amc-N-sucOf-Fe (PSMA-DFO). None of the animals exhibited any pathological signs or clinical symptoms attributable to administration of the PSMA precursor over a 14-day observation period. There were no fatalities from a single dose of 1 mg/kg body weight, and the examination of the organs for visible changes yielded no significant findings. As a result, it was concluded that the lethal dose (LD50) in mice exceeds 1 mg/kg body weight.
 
 

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