GMP production of [18F]FE-PE2I on a TRACERLab FX2 N synthesis module, a radiotracer for in vivo PET imaging of the dopamine transport.

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.

Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer.

BACKGROUND: The Affibody molecule, ABY-025, has demonstrated utility to detect human epidermal growth factor receptor 2 (HER2) in vivo, either radiolabelled with indium-111 (111In) or gallium-68 (68Ga). Using the latter, 68Ga, is preferred due to its use in positron emission tomography with superior resolution and quantifying capabilities in the clinical setting compared to 111In. For an ongoing phase II study (NCT05619016) evaluating ABY-025 for detecting HER2-low lesions and selection of patients for HER2-targeted treatment, the aim was to optimize an automated and cGMP-compliant radiosynthesis of [68Ga]Ga-ABY-025. [68Ga]Ga-ABY-025 was produced on a synthesis module, Modular-Lab PharmTracer (Eckert & Ziegler), commonly used for 68Ga-labelings. The radiotracer has previously been radiolabeled on this module, but to streamline the production, the method was optimized. Steps requiring manual interactions to the radiolabeling procedure were minimized including a convenient and automated pre-concentration of the 68Ga-eluate and a simplified automated final formulation procedure. Every part of the radiopharmaceutical production was carefully developed to gain robustness and to avoid any operator bound variations to the manufacturing. The optimized production method was successfully applied for 68Ga-labeling of another radiotracer, verifying its versatility as a universal and robust method for radiosynthesis of Affibody-based peptides. RESULTS: A simplified and optimized automated cGMP-compliant radiosynthesis method of [68Ga]Ga-ABY-025 was developed. With a decay corrected radiochemical yield of 44 ± 2%, a radiochemical purity (RCP) of 98 ± 1%, and with an RCP stability of 98 ± 1% at 2 h after production, the method was found highly reproducible. The production method also showed comparable results when implemented for radiolabeling another similar peptide. CONCLUSION: The improvements made for the radiosynthesis of [68Ga]Ga-ABY-025, including introducing a pre-concentration of the 68Ga-eluate, aimed to utilize the full potential of the 68Ge/68Ga generator radioactivity output, thereby reducing radioactivity wastage. Furthermore, reducing the number of manually performed preparative steps prior to the radiosynthesis, not only minimized the risk of potential human/operator errors but also enhanced the process' robustness. The successful application of this optimized radiosynthesis method to another similar peptide underscores its versatility, suggesting that our method can be adopted for 68Ga-labeling radiotracers based on Affibody molecules in general. TRIAL REGISTRATION: NCT, NCT05619016, Registered 7 November 2022, https://clinicaltrials.gov/study/NCT05619016?term=HER2&cond=ABY025&rank=1.

The α-synuclein PET tracer [18F] ACI-12589 distinguishes multiple system atrophy from other neurodegenerative diseases.

A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson's disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.

Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging.

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines-one of the most promising structures for in vivo pretargeted applications-were inaccessible using this strategy. We believed that our successful efforts to 18F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [18F]45, which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (Am = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [18F]45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based 'click-to-release' reactions. Consequently, 18F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging-Expanding the Bioorthogonal Tool Box.

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positron-emission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [18F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratios-as early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Insights into Elution of Anion Exchange Cartridges: Opening the Path toward Aliphatic 18F-Radiolabeling of Base-Sensitive Tracers.

Aliphatic nucleophilic substitution (SN2) with [18F]fluoride is the most widely applied method to prepare 18F-labeled positron emission tomography (PET) tracers. Strong basic conditions commonly used during 18F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [18F]fluoride on anion exchange cartridges and elute it afterward with basic anions. This sequence is used to facilitate the transfer of [18F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for SN2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [18O]water from which [18F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits SN2 18F-labeling of base-sensitive scaffolds. Extensive screening of trapping and elution conditions (>1000 experiments) and studying their influence on the radiochemical yield (RCY) allowed us to identify a suitable procedure for this. Using this procedure, four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold) compared to those of previously published procedures, even from lower precursor amounts. Encouraged by these results, we applied our low-basicity method to the radiolabeling of highly base-sensitive tetrazines, which cannot be labeled using state-of-art direct aliphatic 18F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path toward simple and direct SN2 18F fluorination of base-sensitive substrates.

Fully Automated GMP-Compliant Synthesis of [18F]FE-PE2I.

In the struggle to understand and accurately diagnose Parkinson's disease, radiopharmaceuticals and medical imaging techniques have played a major role. By being able to image and quantify the dopamine transporter density, noninvasive diagnostic imaging has become the gold standard. In the shift from the first generation of SPECT tracers, the fluorine-18-labeled tracer [18F]FE-PE2I has emerged as the agent of choice for many physicians. However, implementing suitable synthesis for the production of [18F]FE-PE2I has proved more challenging than expected. Through a thorough analysis of the relevant factors affecting the final radiochemical yield, we were able to implement high-yielding fully automated GMP-compliant synthesis of [18F]FE-PE2I on a Synthera®+ platform. By reaching RCYs up to 62%, it allowed us to isolate 25 GBq of the formulated product, and an optimized formulation resulted in the shelf life of 6 h, satisfying the increased demand for this radiopharmaceutical.

Aliphatic 18 F-Radiofluorination: Recent Advances in the Labeling of Base-Sensitive Substrates*.

Aliphatic fluorine-18 radiolabeling is the most commonly used method to synthesize tracers for PET-imaging. With an increasing demand for 18 F-radiotracers for clinical applications, new labeling strategies aiming to increase radiochemical yields of established tracers or, more importantly, to enable 18 F-labeling of new scaffolds have been developed. In recent years, increased attention has been focused on the direct aliphatic 18 F-fluorination of base-sensitive substrates in this respect. This minireview gives a concise overview of the recent advances within this field and aims to highlight the advantages and limitations of these methods.

Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

In Vivo Veritas: 18F-Radiolabeled Glycomimetics Allow Insights into the Pharmacological Fate of Galectin-3 Inhibitors.

Glycomimetic drugs have attracted increasing interest as unique targeting vectors or surrogates for endogenous biomolecules. However, it is generally difficult to determine the in vivo pharmacokinetic profile of these compounds. In this work, two galectin-3 inhibitors were radiolabeled with fluorine-18 and used as surrogate PET tracers of TD139 and GB1107. Both compounds are promising drugs for clinical applications. In vivo evaluation revealed that both surrogates strongly differed with respect to their biodistribution profile. The disaccharide (TD139 surrogate) was rapidly eliminated from blood while the monosaccharide (GB1107 surrogate) showed no sign of excretion. The data obtained allowed us to infer the different in vivo fate of TD139 and GB1107 and rationalize how different administration routes could boost efficacy. Whereas the fast excretion profile of the TD139 surrogate indicated that systemic application of disaccharides is unfavorable, the extended biological half-life of the GB1107 surrogate indicated that systemic administration is possible for monosaccharides.

Quantitative Mass Spectrometry To Study Inflammatory Cartilage Degradation and Resulting Interactions with the Complement System.

Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period.