Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA

Purpose The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [68Ga]Ga-OncoFAP-DOTAGA (68Ga-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. Methods 68Ga-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of 68Ga-OncoFAP were assessed by determining logD7.4, IC50 values, and radiochemical purity. 68Ga-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 ± 50 MBq 68Ga-OncoFAP combined with PET/CT and PET/MRI. Results 68Ga-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of 68Ga-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting–based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 ± 5.1 at 1 h and 38.1 ± 33.1 at 3 h p.i. Clinical 68Ga-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 ± 2.3), lymph nodes (SUVmax 9.7 ± 8.3), and distant metastases (SUVmax up to 20.0). Conclusion Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate 68Ga-OncoFAP as a powerful alternative to currently available FAP tracers.

Optimization and Evaluation of Al18F Labeling Using a NOTA—or RESCA1-Conjugated AE105 Peptide Antagonist of uPAR

Fluorine-18 displays almost ideal decay properties for positron emission tomography (PET) and allows for large scale production. As such, simplified methods to radiolabel peptides with fluorine-18 are highly warranted. Chelation of aluminium fluoride-18 toward specific peptides represents one method to achieve this. With the current methods, chelation of aluminium fluoride-18 can be achieved using NOTA-conjugated peptides. However, the heating to 90–100◦C that is required for this chelation approach may be deleterious to the targeting moiety of the probe. Recently, a new chelator, RESCA1, was developed allowing Al18F chelation at room temperature. Here, we optimize the labeling procedure enabling high chelation efficacy of fluoride-18 at 22◦C, even at full batch labeling. The optimized procedure was tested by Al18F-labeling of RESCA1-AE105—a uPAR targeting peptide. NOTA-AE105 was also labeled with Al18F, and the two peptides were compared head-to-head. [18F]AlF-NOTA-AE105 and [18F]AlF-RESCA1-AE105 could be produced in equal radiochemical yields (RCY), radiochemical purities (RCP) and molar activities. Additionally, the two peptides showed comparable binding affinity to uPAR and uptake in cells expressing the uPAR, when evaluated in vitro. Overall, we found that the performances of [18F]AlF-NOTA-AE105 and [18F]AlF-RESCA1-AE105 were grossly comparable, but importantly RESCA1 can be labeled with aluminium fluoride-18 at 22◦C. Consequently, this study showed that RESCA1 is superior to NOTA with respect to Al18F chelation of temperature sensitive molecules, such as thermolabile peptides and proteins as well as that full batch chelation of RESCA1 with fluoride-18 is possible.

Microwave-assisted radiolabelling of 52Mn-porphyrins

Manganese porphyrins have several therapeutic/imaging applications; including their use as radioprotectants (in clinical trials), and as paramagnetic MRI contrast agents. The affinity of porphyrins for lipid bilayers also makes them candidates for cell/liposome labelling. We hypothesised that metalation with the positron emission tomography (PET) radionuclide 52Mn (t1/2 = 5.6 d) would allow long-term in vivo biodistribution studies of Mn-porphyrins as well as a method to label and track cells/liposomes, but methods for fast and efficient radiolabelling are lacking. Several porphyrins were produced and radiolabelled by addition to neutralised [52Mn]MnCl2 and heated at 165 oC for 1 h using a microwave (MW) synthesiser at a ligand concentration of 0.6 – 0.7 mM. These conditions were compared with non-MW heating at 70oC. MW radiosynthesis allowed >95 % radiochemical yields (RCY) in just 1 h. Conversely, non-MW heating at 70 oC for 1 h resulted in low RCY (0 – 25 % RCY) and most porphyrins did not reach completion after 24h. Formation of the 52Mn-complexes were confirmed with radio-HPLC by comparison with their non-radioactive 55Mn counterparts. Following this, several 52Mn-porphyrins were used to radiolabel liposomes by incubation at 50 oC for 30 min resulting in 75 – 86 % labelling efficiency (LE). Two lead 52Mn-porphyrins were taken forward to label MDA-MB-231 cancer cells in vitro, achieving ca. 11 % LE. After 24 h, 32 – 45 % of the 52Mn-porphyrin was retained in cells. In contrast to standard methods, MW heating allows fast synthesis of 52Mn-porphyrins with >95% radiochemical yields that avoid purification. 52Mn-porphyrins also show promising cell/liposome labelling properties. This technique can potentially be exploited for the in vivo imaging of Mn-porphyrin therapeutics, as well as for the accurate in vivo quantification of Mn-porphyrin MRI agents.

Radiosynthesis of [11C]Ibrutinib via Pd-Mediated [11C]CO Carbonylation: Preliminary PET Imaging in Experimental Autoimmune Encephalomyelitis Mice

Ibrutinib is a first-generation Bruton's tyrosine kinase (BTK) inhibitor that has shown efficacy in autoimmune diseases and has consequently been developed as a positron emission tomography (PET) radiotracer. Herein, we report the automated radiosynthesis of [11C]ibrutinib through 11C-carbonylation of the acrylamide functional group, by reaction of the secondary amine precursor with [11C]CO, iodoethylene, and palladium–NiXantphos. [11C]Ibrutinib was reliably formulated in radiochemical yields of 5.4% ± 2.5% (non-decay corrected; n = 9, relative to starting [11C]CO2), radiochemical purity >99%, and molar activity of 58.8 ± 30.8 GBq/μmol (1.55 ± 0.83 Ci/μmol). Preliminary PET/magnetic resonance imaging with [11C]ibrutinib in experimental autoimmune encephalomyelitis (EAE) mice showed a 49% higher radioactivity accumulation in the spinal cord of mice with EAE scores of 2.5 vs. sham mice.

MicroPET Imaging of Bacterial Infection with Specifically Nitroreductase Responsive 18F-labeled Nitrogen Mustard Analogues

Purpose Bacterial infection and antibiotic-resistant are still serious threats to human health. Here, we aim to develop two novel radiotracers 18F-NTRP and 18F-NCRP with specific nitroreductase (NTR) responsive to image the deep-seated bacterial infection with PET for differentiating infection from sterile inflammation. Methods 18F-NTRP and 18F-NCRP were synthesized via a one-step method. All the steps of tracers radiosynthesis were successfully adapted in the All-In-One automated module. After the physiochemical properties of 18F-NTRP and 18F-NCRP were characterized, their specificity and selectivity to NTR were verified in E. coli and S. aureus as well. The ex vivo biodistribution of tracers was evaluated in normal mice. MicroPET-CT imaging was acquired in mouse models of bacterial infection and inflammation after the administration of 18F-NTRP and 18F-NCRP. Results Fully automated radiosynthesis of 18F-NTRP and 18F-NCRP were achieved within 90 - 110 min with overall decay-uncorrected isolated radiochemical yields of 21.24 ± 4.25 % and 11.3 ± 3.78 % respectively. Molar activities of 18F-NTRP and 18F-NCRP were 320 ± 40 GBq/µmol and 275 ± 33 GBq/µmol respectively. In addition, 18F-NTRP and 18F-NCRP exhibited good selectivity and specificity to NTR response. PET-CT imaging in bacterial infected mice models with 18F-NTRP and 18F-NCRP showed significant radioactivity uptakes in both E. coli and S. aureus infected muscles. Especially, the E. coli infected muscle uptakes were up to 2.4 ± 0.2 %ID/g (18F-NTRP) and 4.05 ± 0.49 %ID/g (18F-NCRP), 3-fold to control uptake. Furthermore, both 18F-NTRP and 18F-NCRP showed a 2.6-fold higher uptake in bacterial infection compared to inflammation counterparts, indicating that they can distinguish infection from inflammation effectively. Conclusion Based on the promising results of this preliminary study, 18F-NTRP and 18F-NCRP are worth further study to verify their potentials for bacterial infection imaging via NTR responsive.

18F-Fluorination Using Tri-Tert-Butanol Ammonium Iodide as Phase-Transfer Catalyst: An Alternative Minimalist Approach

The 18F syntheses of tracers for positron emission tomography (PET) typically require several steps, including extraction of [18F]fluoride from H2[18O]O, elution, and drying, prior to nucleophilic substitution reaction, being a laborious and time-consuming process. The elution of [18F]fluoride is commonly achieved by phase transfer catalysts (PTC) in aqueous solution, which makes azeotropic drying indispensable. The ideal PTC is characterized by a slightly basic nature, its capacity to elute [18F]fluoride with anhydrous solvents, and its efficient complex formation with [18F]fluoride during subsequent labeling. Herein, we developed tri-(tert-butanol)-methylammonium iodide (TBMA-I), a quaternary ammonium salt serving as the PTC for 18F-fluorination reactions. The favorable elution efficiency of [18F]fluoride using TBMA-I was demonstrated with aprotic and protic solvents, maintaining high 18F-recoveries of 96–99%. 18F-labeling reactions using TBMA-I as PTC were studied with aliphatic 1,3-ditosylpropane and aryl pinacol boronate esters as precursors, providing 18F-labeled products in moderate-to-high radiochemical yields. TBMA-I revealed adequate properties for application to 18F-fluorination reactions and could be used for elution of [18F]fluoride with MeOH, omitting an additional base and azeotropic drying prior to 18F-labeling. We speculate that the tert-alcohol functionality of TBMA-I promotes intermolecular hydrogen bonding, which enhances the elution efficiency and stability of [18F]fluoride during nucleophilic 18F-fluorination.

Radiolabeling of Human Serum Albumin With Terbium-161 Using Mild Conditions and Evaluation of in vivo Stability

Targeted radionuclide therapy (TRNT) is a promising approach for cancer therapy. Terbium has four medically interesting isotopes (149Tb, 152Tb, 155Tb and 161Tb) which span the entire radiopharmaceutical space (TRNT, PET and SPECT imaging). Since the same element is used, accessing the various diagnostic or therapeutic properties without changing radiochemical procedures and pharmacokinetic properties is advantageous. The use of (heat-sensitive) biomolecules as vector molecule with high affinity and selectivity for a certain molecular target is promising. However, mild radiolabeling conditions are required to prevent thermal degradation of the biomolecule. Herein, we report the evaluation of potential bifunctional chelators for Tb-labeling of heat-sensitive biomolecules using human serum albumin (HSA) to assess the in vivo stability of the constructs. p-SCN-Bn-CHX-A”-DTPA, p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA were conjugated to HSA via a lysine coupling method. All HSA-constructs were labeled with [161Tb]TbCl3 at 40°C with radiochemical yields higher than 98%. The radiolabeled constructs were stable in human serum up to 24 h at 37°C. 161Tb-HSA-constructs were injected in mice to evaluate their in vivo stability. Increasing bone accumulation as a function of time was observed for [161Tb]TbCl3 and [161Tb]Tb-DTPA-CHX-A”-Bn-HSA, while negligible bone uptake was observed with the DOTA, DOTA-GA and NETA variants over a 7-day period. The results indicate that the p-SCN-Bn-DOTA, p-NCS-Bz-DOTA-GA and p-SCN-3p-C-NETA are suitable bifunctional ligands for Tb-based radiopharmaceuticals, allowing for high yield radiolabeling in mild conditions.

Re-Evaluations of Zr-DFO Complex Coordination Chemistry for the Estimation of Radiochemical Yields and Chelator-to-Antibody Ratios of 89Zr Immune-PET Tracers

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logβ111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

GMP-Compliant Radiosynthesis of [18F]GP1, a Novel PET Tracer for the Detection of Thrombi

Thrombus formation and thromboembolic events play important roles in various cardiovascular pathologies. The key receptor involved in platelet aggregation is the fibrinogen receptor glycoprotein IIb/IIIa. [18F]GP1, a derivative of the GPIIb/IIIa antagonist elarofiban, is a specific 18F-labeled small-molecule radiotracer that binds with high affinity to GPIIb/IIIa receptors of activated platelets. An improved, robust and fully automated radiosynthesis of [18F]GP1 has been developed. [18F]GP1 has been synthesized with decay corrected radiochemical yields of 38 ± 6%, with a radiochemical concentration up to 1900 MBq/mL, molar activities of 952–9428 GBq/µmol and a radio-chemical purity >98%. After determination of the optimal reaction conditions, in particular for HPLC separation, adaption of the reaction conditions to PET center requirements, validation of the manufacturing process and the quality control methods, the synthesis of [18F]GP1 was successfully implemented to GMP standards and was available for clinical application. We describe the GMP-compliant synthesis of the novel radiotracer [18F]GP1. Moreover, we provide some proof-of-concept examples for clinical application in the cardiovascular field. PET/CT with the novel small-molecular radiotracer [18F]GP1 may serve as a novel highly sensitive tool for visualizing active platelet aggregation at the molecular level.