EJNMMI Radiopharmacy and Chemistry最新文献

Background

During the previous two decades, PET imaging of biopharmaceuticals radiolabeled with zirconium-89 has become a consistent tool in preclinical and clinical drug development and patient selection, primarily due to its advantageous physical properties that allow straightforward radiolabeling of antibodies (⁸⁹Zr-immuno-PET). The extended half-life of 78.4 h permits flexibility with respect to the logistics of tracer production, transportation, and imaging and allows imaging at later points in time. Additionally, its relatively low positron energy contributes to high-sensitivity, high-resolution PET imaging. Considering the growing interest in radiolabeling antibodies, antibody derivatives, and other compound classes with ⁸⁹Zr in both clinical and pre-clinical settings, there is an urgent need to acquire valuable recommendations and guidelines towards standardization of labeling procedures.

Main body

This review provides an overview of the key aspects of ⁸⁹Zr-radiochemistry and radiopharmaceuticals. Production of ⁸⁹Zr, conjugation with the mostly used chelators and radiolabeling strategies, and quality control of the radiolabeled products are described in detail, together with discussions about alternative options and critical steps, as well as recommendations for troubleshooting. Moreover, some historical background on ⁸⁹Zr-immuno-PET, coordination chemistry of ⁸⁹Zr, and future perspectives are provided. This review aims to serve as a quick-start guide for scientists new to the field of ⁸⁹Zr-immuno-PET and to suggest approaches for harmonization and standardization of current procedures.

Conclusion

The favorable PET imaging characteristics of ⁸⁹Zr, its excellent availability due to relatively simple production and purification processes, and the development of suitable bifunctional chelators have led to the widespread use of ⁸⁹Zr. The combination of antibodies and ⁸⁹Zr, known as ⁸⁹Zr-immuno-PET, has become a cornerstone in drug development and patient selection in recent years. Despite the advanced state of ⁸⁹Zr-immuno-PET, new developments in chelator conjugation and radiolabeling procedures, application in novel compound classes, and improved PET scanner technology and quantification methods continue to reshape its landscape towards improving clinical outcomes.

Background

Automated [⁸⁹Zr]Zr-radiolabeling processes have the potential to streamline the production of [⁸⁹Zr]Zr-labelled PET imaging agents. Most radiolabeling protocols use [⁸⁹Zr][Zr(ox)₄]⁴⁻ as the starting material and oxalate is removed after radiolabeling. In some instances, radiolabeling with [⁸⁹Zr]ZrCl₄ as starting material gives better radiochemical yields at lower reaction temperatures. In this work, a fully-automated process for production of [⁸⁹Zr]ZrCl₄ is reported and its use for the synthesis of [⁸⁹Zr]ZrDFOSq-bisPhPSMA and [⁸⁹Zr]ZrDFOSq-TATE.

Results

A simple automated process for the isolation of [⁸⁹Zr]ZrCl₄ by trapping [⁸⁹Zr][Zr(ox)₄]⁴⁻ on a bicarbonate-activated strong anion exchange cartridge followed by elution with 0.1 M HCl in 1 M NaCl was developed. [⁸⁹Zr]ZrCl₄ was routinely recovered from [⁸⁹Zr][Zr(ox)₄]⁴⁻ in > 95% yield in mildly acidic solution of 0.1 M HCl in 1 M NaCl using a fully-automated process. The [⁸⁹Zr]ZrCl₄ was neutralized with sodium acetate buffer (0.25 M) removing the requirement for cumbersome manual neutralization with strong base. The mixture of [⁸⁹Zr]ZrCl₄ was used for direct automated radiolabeling reactions to produce [⁸⁹Zr]Zr-DFOSquaramide-bisPhPSMA and [⁸⁹Zr]ZrDFOSquaramide-TATE in 80–90% over all RCY in > 95% RCP.

Conclusions

This method for the production of [⁸⁹Zr]ZrCl₄ does not require removal of HCl by evaporation making this process relatively fast and efficient. The fully automated procedures for the production of [⁸⁹Zr]ZrCl₄ and its use in radiolabeling are well suited to support the centralized and standardized manufacture of multiple dose preparations of zirconium-89 based radiopharmaceuticals.

Background

Positron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (⁸⁹Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, ⁸⁹Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs.

Results

The main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable ⁸⁹Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective ⁸⁹Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with ⁸⁹Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe³⁺, Zn²⁺ and Cu²⁺. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry.

Conclusion

Combining solid phase and solution synthesis techniques, we identified novel ⁸⁹Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of ⁸⁹Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.

Background

Parkinson's disease is a neurodegenerative disorder that is characterized by a degeneration of the dopaminergic system. Dopamine transporter (DAT) positron emission tomography (PET) imaging has emerged as a powerful and non-invasive method to quantify dopaminergic function in the living brain. The PET radioligand, [¹⁸F]FE-PE2I, a cocaine chemical derivative, has shown promising properties for in vivo PET imaging of DAT, including high affinity and selectivity for DAT, excellent brain permeability, and favorable metabolism. The aim of the current study was to scale up the production of [¹⁸F]FE-PE2I to fulfil the increasing clinical demand for this tracer.

Results

Thus, a fully automated and GMP-compliant production procedure has been developed using a commercially available radiosynthesis module GE TRACERLab FX2 N. [¹⁸F]FE-PE2I was produced with a radiochemical yield of 39 ± 8% (n = 4, relative [¹⁸F]F⁻ delivered to the module). The synthesis time was 70 min, and the molar activity was 925.3 ± 763 GBq/µmol (250 ± 20 Ci/µmol). The produced [¹⁸F]FE-PE2I was stable over 6 h at room temperature.

Conclusion

The protocol reliably provides a sterile and pyrogen–free GMP-compliant product.

Background

In recent years, fibroblast activating protein (FAP), a biomarker overexpressed by cancer-associated fibroblasts, has emerged as one of the most promising biomarkers in oncology. Similarly, FAP overexpression has been detected in various fibroblast-mediated inflammatory conditions such as liver cirrhosis and idiopathic pulmonary fibrosis. Along this trajectory, FAP-targeted positron emission tomography (PET), utilizing FAP inhibitors (FAPi) labeled with positron emitters, has gained traction as a powerful imaging approach in both cancer and inflammation. However, PET represents a high-cost technology, and its widespread adoption is still limited compared to the availability of gamma cameras. To address this issue, several efforts have been made to explore the potential of [^99mTc]Tc-FAPi tracers as molecular probes for imaging with gamma cameras and single photon emission computed tomography (SPECT).

Main body

Several approaches have been investigated for labeling FAPi-based compounds with ^99mTc. Specifically, the mono-oxo, tricarbonyl, isonitrile, and HYNIC strategies have been applied to produce [^99mTc]Tc-FAPi tracers, which have been tested in vitro and in animal models. Overall, these labeling approaches have demonstrated high efficiency and strong binding. The resulting [^99mTc]Tc-FAPi tracers have shown high specificity for FAP-positive cells and xenografts in both in vitro and animal model studies, respectively. However, the majority of [^99mTc]Tc-FAPi tracers have exhibited variable levels of lipophilicity, leading to preferential excretion through the hepatobiliary route and undesirable binding to lipoproteins. Consequently, efforts have been made to synthesize more hydrophilic FAPi-based compounds to improve pharmacokinetic properties and achieve a more favorable biodistribution, particularly in the abdominal region. SPECT imaging with [^99mTc]Tc-FAPi has yielded promising results in patients with gastrointestinal tumors, demonstrating comparable or superior diagnostic performance compared to other imaging modalities. Similarly, encouraging outcomes have been observed in subjects with gliomas, lung cancer, breast cancer, and cervical cancer. Beyond oncological applications, [^99mTc]Tc-FAPi-based imaging has been successfully employed in myocardial and idiopathic pulmonary fibrosis.

Conclusions

This overview focuses on the various radiochemical strategies for obtaining [^99mTc]Tc-FAPi tracers, highlighting the main challenges encountered and possible solutions when applying each distinct approach. Additionally, it covers the preclinical and initial clinical applications of [^99mTc]Tc-FAPi in cancer and inflammation.

Background

6-Bromo-7-[¹¹C]methylpurine ([¹¹C]BMP) is a radiotracer for positron emission tomography (PET) to measure multidrug resistance-associated protein 1 (MRP1) transport activity in different tissues. Previously reported radiosyntheses of [¹¹C]BMP afforded a mixture of 7- and 9-[¹¹C]methyl regioisomers. To prepare for clinical use, we here report an improved regioselective radiosynthesis of [¹¹C]BMP, the results of a non-clinical toxicity study as well as human dosimetry estimates based on mouse PET data.

Results

[¹¹C]BMP was synthesised by regioselective N⁷-methylation of 6-bromo-7H-purine (prepared under good manufacturing practice) with [¹¹C]methyl triflate in presence of 2,2,6,6-tetramethylpiperidine magnesium chloride in a TRACERlab™ FX2 C synthesis module. [¹¹C]BMP was obtained within a total synthesis time of approximately 43 min in a decay-corrected radiochemical yield of 20.5 ± 5.2%, based on starting [¹¹C]methyl iodide, with a radiochemical purity > 99% and a molar activity at end of synthesis of 197 ± 130 GBq/μmol (n = 28). An extended single-dose toxicity study conducted in male and female Wistar rats under good laboratory practice after single intravenous (i.v.) administration of unlabelled BMP (2 mg/kg body weight) revealed no test item related adverse effects. Human dosimetry estimates, based on dynamic whole-body PET data in female C57BL/6J mice, suggested that an i.v. injected activity amount of 400 MBq of [¹¹C]BMP will deliver an effective dose in the typical range of ¹¹C-labelled radiotracers.

Conclusions

[¹¹C]BMP can be produced in sufficient amounts and acceptable quality for clinical use. Data from the non-clinical safety evaluation showed no adverse effects and suggested that the administration of [¹¹C]BMP will be safe and well tolerated in humans.

Background

The aim of this study was to prepare a novel ⁶⁸Ga-labeled pH (low) insertion peptide (pHLIP)-like peptide, YJL-4, and determine its value for the early diagnosis of triple-negative breast cancer (TNBC) via in vivo imaging of tumor-bearing nude mice. The novel peptide YJL-4 was designed using a template-assisted method and synthesized by solid-phase peptide synthesis. After modification with the chelator 1,4,7‑triazacyclononane-N,N′,N″-triacetic acid (NOTA), the peptide was labeled with ⁶⁸Ga. Then, the biodistribution of ⁶⁸Ga-YJL-4 in tumor-bearing nude mice was investigated, and the mice were imaged by small animal positron emission tomography (PET).

Results

The radiochemical yield and radiochemical purity of ⁶⁸Ga-YJL-4 were 89.5 ± 0.16% and 97.95 ± 0.06%, respectively. The biodistribution of ⁶⁸Ga-YJL-4 in tumors (5.94 ± 1.27% ID/g, 6.72 ± 1.69% ID/g and 4.54 ± 0.58% ID/g at 1, 2 and 4 h after injection, respectively) was significantly greater than that of the control peptide in tumors at the corresponding time points (P < 0.01). Of the measured off-target organs, ⁶⁸Ga-YJL-4 was highly distributed in the liver and blood. The small animal PET imaging results were consistent with the biodistribution results. The tumors were visualized by PET at 2 and 4 h after the injection of ⁶⁸Ga-YJL-4. No tumors were observed in the control group.

Conclusions

The novel pHLIP family peptide YJL-4 can adopt an α-helical structure for easy insertion into the cell membrane in an acidic environment. ⁶⁸Ga-YJL-4 was produced in high radiochemical yield with good stability and can target TNBC tissue. Moreover, the strong concentration of radioactive ⁶⁸Ga-YJL-4 in the abdomen does not hinder the imaging of early TNBC.

Background

Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of ⁶⁸Ga- and ^99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics.

Results

The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both ⁶⁸Ga- and ^99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described.

Conclusion

The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

Graphical abstract

Background

Steps have been taken by pharmaceutical companies to obtain marketing authorisation of PSMA ligands in the European Union. Since December 2022, Locametz® (PSMA-11, gozetotide) is licensed as kit for manual radiolabelling with gallium-68 and commercially available since mid-2023. The Summary of Product Characteristic (SmPC) describes manual radiolabelling with a maximum activity after radiolabelling of 1369 MBq. We aimed for radiolabelling with a higher activity to increase production efficiency, and thus, automated radiolabelling is strongly preferred over manual radiolabelling to reduce radiation exposure to personnel. The aim of this study was to develop and validate a method for automated radiolabelling of the Locametz® kit using ~ 2000 MBq of gallium-68 eluate for radiolabelling.

Results

Automated radiolabelling of [⁶⁸Ga]Ga-PSMA-11 using the Locametz® kit provided a product which complies to the Ph. Eur., had a shelf-life of 6 h at room temperature, and theoretically reduced radiation exposure 5.7 times. Radiolabelling with one and two generator(s) resulted in a radiochemical yield of 91–102% and 96–101% after preparation, respectively. The radiochemical purity ranged from 98.0 to 99.6% for radiolabelling with one generator and ranged from 98.4 to 99.3% for radiolabelling with two generators with similar stability. The activity of the final product was much higher when using two generators, 1961–2035 MBq compared to 740–1260 MBq, which leads to ~ 1.5 times more patient syringes available per preparation.

Conclusion

Automated radiolabelling of [⁶⁸Ga]Ga-PSMA-11 using the Locametz® kit with higher gallium-68 activity than specified in the SmPC results in a product that is in compliance with the Ph. Eur. monograph and has a shelf-life of 6 h at room temperature. Radiolabelling with two generators proved possible and resulted in a product with similar quality but with much higher efficiency.