Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

Hetero-Diels-Alder click reaction of dithioesters for a catalyst-free indirect 18F-radiolabelling of peptides.

The HDA reaction of dithioesters was developed as a new click-reaction compatible with the indirect 18F-labelling of peptides. It involves dithioester-peptides and a radiofluorinated diene as a novel prosthetic group. The method was applied to a PSMA-ligand for the in vivo detection of LNCap tumors in xenografted mice.

64CuCl2 PET Imaging of 4T1-Related Allograft of Triple-Negative Breast Cancer in Mice.

64CuCl2 is an economic radiotracer for oncologic PET investigations. In the present study, we characterized the uptake of 64CuCl2 in vivo by µPET/CT in an allograft 4T1-related mouse model (BALB/c) of advanced breast cancer. 18F-FDG was used as a comparator. Twenty-two animals were imaged 7-9 days following 4T1-cell implantation inside mammary glands. Dynamic 64CuCl2 µPET/CT acquisition or iterative static images up to 8 h p.i. were performed. Animal biodistribution and tumor uptake were first evaluated in vivo by µPET analysis and then assessed on tissue specimens. Concerning 18F-FDG µPET, a static acquisition was performed at 15 min and 60 min p.i. Tumor 64CuCl2 accumulation increased from 5 min to 4 h p.i., reaching a maximum value of 5.0 ± 0.20 %ID/g. Liver, brain, and muscle 64CuCl2 accumulation was stable over time. The tumor-to-muscle ratio remained stable from 1 to 8 h p.i., ranging from 3.0 to 3.7. Ex vivo data were consistent with in vivo estimations. The 18F-FDG tumor accumulation was 8.82 ± 1.03 %ID/g, and the tumor-to-muscle ratio was 4.54 ± 1.11. 64CuCl2 PET/CT provides good characterization of the 4T1-related breast cancer model and allows for exploration of non-glycolytic cellular pathways potentially of interest for theragnostic strategies.

Performance evaluation of the IRIS XL-220 PET/CT system, a new camera dedicated to non-human primates.

BACKGROUND: Non-human primates (NHP) are critical in biomedical research to better understand the pathophysiology of diseases and develop new therapies. Based on its translational and longitudinal abilities along with its non-invasiveness, PET/CT systems dedicated to non-human primates can play an important role for future discoveries in medical research. The aim of this study was to evaluate the performance of a new PET/CT system dedicated to NHP imaging, the IRIS XL-220 developed by Inviscan SAS. This was performed based on the National Electrical Manufacturers Association (NEMA) NU 4-2008 standard recommendations (NEMA) to characterize the spatial resolution, the scatter fraction, the sensitivity, the count rate, and the image quality of the system. Besides, the system was evaluated in real conditions with two NHP with 18F-FDG and (-)-[18F]FEOBV which targets the vesicular acetylcholine transporter, and one rat using 18F-FDG. RESULTS: The full width at half maximum obtained with the 3D OSEM algorithm ranged between 0.89 and 2.11 mm in the field of view. Maximum sensitivity in the 400-620 keV and 250-750 keV energy windows were 2.37% (22 cps/kBq) and 2.81% (25 cps/kBq), respectively. The maximum noise equivalent count rate (NEC) for a rat phantom was 82 kcps at 75 MBq and 88 kcps at 75 MBq for energy window of 250-750 and 400-620 keV, respectively. For the monkey phantom, the maximum NEC was 18 kcps at 126 MBq and 19 kcps at 126 MBq for energy window of 250-750 and 400-620 keV, respectively. The IRIS XL provided an excellent quality of images in non-human primates and rats using 18F-FDG. The images acquired using (-)-[18F]FEOBV were consistent with those previously reported in non-human primates. CONCLUSIONS: Taken together, these results showed that the IRIS XL-220 is a high-resolution system well suited for PET/CT imaging in non-human primates.

Comparison of the [18F]-FDG and [18F]-FLT PET Tracers in the Evaluation of the Preclinical Proton Therapy Response in Hepatocellular Carcinoma.

PURPOSE: The main objective of the present study was to compare the 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) and 3'-[18F]fluoro-3'-deoxythymidine ([18F]-FLT) PET imaging biomarkers for the longitudinal follow-up of small animal proton therapy studies in the context of hepatocellular carcinoma (HCC). PROCEDURES: SK-HEP-1 cells were injected into NMRI nude mice to mimic human HCC. The behavior of [18F]-FDG and [18F]-FLT tumor uptake was evaluated after proton therapy procedures. The proton single-fraction doses were 5, 10, and 20 Gy, with a dose rate of 10 Gy/min. The experimental protocol consisted of 8 groups of 10 mice, each group experiencing a particular dose/radiotracer condition. A reference PET exam was performed on each mouse the day before the irradiation procedure, followed by PET exams every 3 days up to 16 days after irradiation. RESULTS: [18F]-FDG uptake showed a linear dose-dependent increase in the first days after treatment (37%, p < 0.05), while [18F]-FLT uptake decreased in a dose-dependent manner (e.g., 21% for 5 Gy compared to 10 Gy, p = 1.1e-2). At the later time point, [18F]-FDG normalized activity showed an 85% decrease (p < 0.01) for both 10 and 20 Gy doses and no variation for 5 Gy. Conversely, a significant 61% (p = 0.002) increase was observed for [18F]-FLT normalized activity at 5 Gy and no variation for higher doses. CONCLUSION: We showed that the use of the [18F]-FDG and [18F]-FLT radiolabeled molecules can provide useful and complementary information for longitudinal follow-up of small animal proton therapy studies in the context of HCC. [18F]-FDG PET imaging enables a treatment monitoring several days/weeks postirradiation. On the other hand, [18F]-FLT could represent a good candidate to monitor the treatment few days postirradiation, in the context of hypo-fractioned and close irradiation planning. This opens new perspectives in terms of treatment efficacy verification depending on the irradiation scheme.

O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) uptake in insulinoma: first results from a xenograft mouse model and from human.

INTRODUCTION: Herein we have evaluated the uptake of O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) in insulinoma in comparison with those of 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-FDOPA) providing first data from both murine xenograft model and one patient with proved endogenous hyperinsulinemic hypoglycemia. METHODS: Dynamic 18F-FET and carbidopa-assisted 18F-FDOPA PET were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F murine beta cells and on a 30-year-old man with type-1 multiple endocrine neoplasia and hyperinsulinemic hypoglycemia defined by a positive fasting test. RESULTS: Seven and three nude mice bearing a RIN-m5F insulinoma xenograft were respectively studied by 18F-FET and 18F-FDOPA µPET. Insulinoma xenograft was detected in all the imaged animals. Xenograft was characterized by an early but moderate increase of 18F-FET uptake followed by a slight decline of uptake intensity during the 20 min dynamic acquisition. Tumoral radiotracer peak intensity and the highest tumor-to-background contrast were reached about 5 minutes after 18F-FET iv. injection (mean SUV: 1.21 ± 0.10). The biodistribution of 18F-FET and 18F-FDOPA and their dynamic tumoral uptake profile and intensity were similar. In the examined patient, 18F-FDOPA and 18F-FET PET/CT showed one concordant focal area of well-defined increased uptake in the pancreatic tail corresponding to 11 mm histologically proved insulinoma. The SUVmax tumor to liver ratio was 1.5, 1.1 for 18F-FDOPA, 1.1, 1 for 18F-FET at early (0-5 min post injection) and delayed (5-20 min post injection) PET/CT acquisition, respectively. Despite the relatively low tumoral uptake intensity, insulinoma was clearly identified due to the low background in the pancreas. At the contrary, no 18F-FDOPA or 18F-FET tumoral uptake was revealed on whole-body PET/CT images performed about 30 min after radiotracer administration. Note of worth, the dynamic uptake pattern of 18F-FET and 18F-FDOPA were similar between human insulinoma and mice xenograft tumor. CONCLUSION: 18F-FET PET compared equally to 18F-FDOPA PET in a preclinical RIN-m5F murine model of insulinoma and in one patient with insulinoma-related hypoglycemia. However, in both cases, the tumoral uptake intensity was moderate and the tumor was only visible until 20 min after radiotracer injection. Hence, caution should be taken before asserting the translational relevance of our results in the clinical practices. However, the structural analogies between 18F-FET and 18F-FDOPA as well as the limited pancreatic uptake of 18F-FET in human, encourage evaluating 18F-FET as diagnostic radiotracer for insulinoma detection in further prospective studies involving large cohorts of patients.

Effect of Carbidopa on 18F-FDOPA Uptake in Insulinoma: From Cell Culture to Small-Animal PET Imaging.

Patient premedication with carbidopa seems to improve the accuracy of 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-FDOPA) PET for insulinoma diagnosis. However, the risk of PET false-negative results in the presence of carbidopa is a concern. Consequently, we aimed to evaluate the effect of carbidopa on 18F-FDOPA uptake in insulinoma ß-cells and an insulinoma xenograft model in mice. METHODS: 18F-FDOPA in vitro accumulation was assessed in the murine ß-cell line RIN-m5F. In vivo small-animal PET experiments were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F cells. Experiments were conducted with and without carbidopa pretreatment. RESULTS: Incubation of RIN-m5F cells with 80 µM carbidopa did not significantly affect the cellular accumulation of 18F-FDOPA. Tumor xenografts were clearly detectable by small-animal PET in all cases. Insulinoma xenografts in carbidopa-treated mice showed significantly higher 18F-FDOPA uptake than those in nontreated mice. Regardless of carbidopa premedication, the xenografts were characterized by an early increase in 18F-FDOPA uptake and then a progressive reduction over time. CONCLUSION: Carbidopa did not influence in vitro 18F-FDOPA accumulation in RIN-m5F cells but improved insulinoma imaging in vivo. Our findings increase current knowledge about the 18F-FDOPA uptake profile of RIN-m5F cells and a related xenograft model. To our knowledge, the present work represents the first preclinical research specifically focused on insulinomas, with potential translational implications.

Automated and efficient radiosynthesis of [(18)F]FLT using a low amount of precursor.

INTRODUCTION: Since 1991 until now, many radiosyntheses of [(18)F]FLT have been published. Most of them suffer from side reactions and/or difficult purification related to the large amount of precursor necessary for the labeling step. A fully automated synthesis using only commercial and unmodified materials with a reduced amount of precursor would be desirable. METHODS: We first explored the possibility to elute efficiently [(18)F]fluorine from commercial and unmodified cartridges with various amount of base. Based on these results, 10mg and 5mg of precursors were used for the fluorination step. The best conditions were transposed in an automated process for a one pot two steps synthesis of labeled FLT. RESULTS: Using commercial and non-treated carbonate form of QMA cartridges, we were able to elute quantitatively the [(18)F]fluorine with a very low amount of base (0.59mg) and, with only 5mg of precursor, to perform an efficient fluorination reaction with up to 94% incorporation of [(18)F]fluorine. The synthesis was fully automated and radiochemical yields of 54% (decay corrected) were obtained within a synthesis time of 52minutes. CONCLUSION: We demonstrate that a fully automated and efficient radiosynthesis of [(18)F]FLT is feasible with only 5mg of precursor. Compare to the present state of the art, our method provides high yields of pure [(18)F]FLT and is broadly adaptable to other synthesis automates.

Simultaneous scanning of two mice in a small-animal PET scanner: a simulation-based assessment of the signal degradation.

In PET imaging, research groups have recently proposed different experimental set ups allowing multiple animals to be simultaneously imaged in a scanner in order to reduce the costs and increase the throughput. In those studies, the technical feasibility was demonstrated and the signal degradation caused by additional mice in the FOV characterized, however, the impact of the signal degradation on the outcome of a PET study has not yet been studied. Here we thoroughly investigated, using Monte Carlo simulated [18F]FDG and [11C]Raclopride PET studies, different experimental designs for whole-body and brain acquisitions of two mice and assessed the actual impact on the detection of biological variations as compared to a single-mouse setting. First, we extended the validation of the PET-SORTEO Monte Carlo simulation platform for the simultaneous simulation of two animals. Then, we designed [18F]FDG and [11C]Raclopride input mouse models for the simulation of realistic whole-body and brain PET studies. Simulated studies allowed us to accurately estimate the differences in detection between single- and dual-mode acquisition settings that are purely the result of having two animals in the FOV. Validation results showed that PET-SORTEO accurately reproduced the spatial resolution and noise degradations that were observed with actual dual phantom experiments. The simulated [18F]FDG whole-body study showed that the resolution loss due to the off-center positioning of the mice was the biggest contributing factor in signal degradation at the pixel level and a minimal inter-animal distance as well as the use of reconstruction methods with resolution modeling should be preferred. Dual mode acquisition did not have a major impact on ROI-based analysis except in situations where uptake values in organs from the same subject were compared. The simulated [11C]Raclopride study however showed that dual-mice imaging strongly reduced the sensitivity to variations when mice were positioned side-by-side while no sensitivity reduction was observed when they were facing each other. This is the first study showing the impact of different experimental designs for whole-body and brain acquisitions of two mice on the quality of the results using Monte Carlo simulated [18F]FDG and [11C]Raclopride PET studies.

4D PET iterative deconvolution with spatiotemporal regularization for quantitative dynamic PET imaging.

Quantitative measurements in dynamic PET imaging are usually limited by the poor counting statistics particularly in short dynamic frames and by the low spatial resolution of the detection system, resulting in partial volume effects (PVEs). In this work, we present a fast and easy to implement method for the restoration of dynamic PET images that have suffered from both PVE and noise degradation. It is based on a weighted least squares iterative deconvolution approach of the dynamic PET image with spatial and temporal regularization. Using simulated dynamic [(11)C] Raclopride PET data with controlled biological variations in the striata between scans, we showed that the restoration method provides images which exhibit less noise and better contrast between emitting structures than the original images. In addition, the method is able to recover the true time activity curve in the striata region with an error below 3% while it was underestimated by more than 20% without correction. As a result, the method improves the accuracy and reduces the variability of the kinetic parameter estimates calculated from the corrected images. More importantly it increases the accuracy (from less than 66% to more than 95%) of measured biological variations as well as their statistical detectivity.

Simulation-based optimisation of the PET data processing for partial saturation approach protocols.

Positron emission tomography (PET) with [(11)C]Raclopride is an important tool for studying dopamine D2 receptor expression in vivo. [(11)C]Raclopride PET binding experiments conducted using the Partial Saturation Approach (PSA) allow the estimation of receptor density (B(avail)) and the in vivo affinity appK(D). The PSA is a simple, single injection, single scan experimental protocol that does not require blood sampling, making it ideal for use in longitudinal studies. In this work, we generated a complete Monte Carlo simulated PET study involving two groups of scans, in between which a biological phenomenon was inferred (a 30% decrease of B(avail)), and used it in order to design an optimal data processing chain for the parameter estimation from PSA data. The impact of spatial smoothing, noise removal and image resolution recovery technique on the statistical detection was investigated in depth. We found that image resolution recovery using iterative deconvolution of the image with the system point spread function associated with temporal data denoising greatly improves the accuracy and the statistical reliability of detecting the imposed phenomenon. Before optimisation, the inferred B(avail) variation between the two groups was underestimated by 42% and detected in 66% of cases, while a false decrease of appK(D) by 13% was detected in more than 11% of cases. After optimisation, the calculated B(avail) variation was underestimated by only 3.7% and detected in 89% of cases, while a false slight increase of appK(D) by 3.7% was detected in only 2% of cases. We found during this investigation that it was essential to adjust a factor that accounts for difference in magnitude between the non-displaceable ligand concentrations measured in the target and in the reference regions, for different data processing pathways as this ratio was affected by different image resolutions.

Imaging capabilities of the Inveon SPECT system using single-and multipinhole collimators.

UNLABELLED: The Inveon small-animal SPECT system comes with several types of multipinhole collimator plates. We evaluate here the performance measurements of the Inveon SPECT system using 6 different collimators: 3 dedicated for mouse imaging and 3 for rat imaging. METHODS: The measured performance parameters include the sensitivity, the spatial resolution using line sources, the ultra-micro Derenzo phantom, the recovery coefficient and the noise measurements using the National Electrical Manufacturers Association NU-4 image quality phantom, obtained with the 2 reconstruction algorithms available with the Inveon Acquisition Workplace, version 1.5-the 3-dimensional ordered-subset expectation maximization (3DOSEM) and the 3-dimensional maximum a posteriori (3DMAP). Further, the overall performance of the system is illustrated by an animal experiment. RESULTS: The results show that the Inveon SPECT scanner offers a spatial resolution, measured at the center of the field of view, ranging from 0.6 to 1 mm with the collimator plates dedicated to mouse imaging and from 1.2 to less than 2 mm with rat collimator plates. The system sensitivity varies from 29 to 404 cps/MBq for mouse collimators and from 53 to 175 cps/MBq for rat collimators. The image quality study showed that 3DMAP allows better noise reduction while preserving the recovery coefficient, compared with other regularization strategies such as the premature termination of the 3DOSEM reconstruction or 3DOSEM followed by gaussian filtering. CONCLUSION: The acquisition parameters, such as the collimator set and the radius of rotation, offer a wide range of possibilities to apply to a large number of biologic studies. However, special care must be taken because this increase in sensitivity can be offset by image degradation, such as image artifacts caused by projection overlap and statistical noise due to a higher number of iterations required for convergence. 3DMAP allowed better noise reduction while maintaining relatively constant recovery coefficients, as compared with other reconstruction strategies.