Hypoxia Imaging Using PET and SPECT: The Effects of Anesthetic and Carrier Gas on [64Cu]-ATSM, [99mTc]-HL91 and [18F]-FMISO Tumor Hypoxia Accumulation

Background

Preclinical imaging requires anaesthesia to reduce motion-related artefacts. For direct translational relevance, anaesthesia must not significantly alter experimental outcome. This study reports on the effects of both anaesthetic and carrier gas upon the uptake of [⁶⁴Cu]-CuATSM, [^99mTc]-HL91 and [¹⁸F]-FMISO in a preclinical model of tumor hypoxia.

Methodology/Principal Findings

The effect of carrier gas and anaesthetic was studied in 6 groups of CaNT-bearing CBA mice using [⁶⁴Cu]-CuATSM, [^99mTc]-HL91 or [¹⁸F]-FMISO. Mice were anaesthetised with isoflurane in air, isoflurane in pure oxygen, with ketamine/xylazine or hypnorm/hypnovel whilst breathing air, or in the awake state whilst breathing air or pure oxygen. PET or SPECT imaging was performed after which the mice were killed for organ/tumor tracer quantitation. Tumor hypoxia was confirmed. Arterial blood gas analysis was performed for the different anaesthetic regimes. The results demonstrate marked influences on tumor uptake of both carrier gas and anaesthetic, and show differences between [^99mTc]-HL91, [¹⁸F]-FMISO and [⁶⁴Cu]-CuATSM. [^99mTc]-HL91 tumor uptake was only altered significantly by administration of 100% oxygen. The latter was not the case for [¹⁸F]-FMISO and [⁶⁴Cu]-CuATSM. Tumor-to-muscle ratio (TMR) for both compounds was reduced significantly when either oxygen or anaesthetics (isoflurane in air, ketamine/xylazine or hypnorm/hypnovel) were introduced. For [¹⁸F]-FMISO no further decrease was measured when both isoflurane and oxygen were administered, [⁶⁴Cu]-CuATSM did show an additional significant decrease in TMR. When using the same anaesthetic regimes, the extent of TMR reduction was less pronounced for [⁶⁴Cu]-CuATSM than for [¹⁸F]-FMISO (40–60% versus 70% reduction as compared to awake animals breathing air).

Conclusions/Significance

The use of anaesthesia can have profound effects on the experimental outcome. More importantly, all tested anaesthetics reduced tumor-hypoxia uptake. Anaesthesia cannot be avoided in preclinical studies but great care has to be taken in preclinical models of hypoxia as anaesthesia effects cannot be generalised across applications, nor disease states.     

Construction and Evaluation of Quantitative Small-Animal PET Probabilistic Atlases for [18F]FDG and [18F]FECT Functional Mapping of the Mouse Brain

Automated voxel-based or pre-defined volume-of-interest (VOI) analysis of small-animal PET data in mice is necessary for optimal information usage as the number of available resolution elements is limited. We have mapped metabolic ([¹⁸F]FDG) and dopamine transporter ([¹⁸F]FECT) small-animal PET data onto a 3D Magnetic Resonance Microscopy (MRM) mouse brain template and aligned them in space to the Paxinos co-ordinate system. In this way, ligand-specific templates for sensitive analysis and accurate anatomical localization were created. Next, using a pre-defined VOI approach, test-retest and intersubject variability of various quantification methods were evaluated. Also, the feasibility of mouse brain statistical parametric mapping (SPM) was explored for [¹⁸F]FDG and [¹⁸F]FECT imaging of 6-hydroxydopamine-lesioned (6-OHDA) mice.

Methods

Twenty-three adult C57BL6 mice were scanned with [¹⁸F]FDG and [¹⁸F]FECT. Registrations and affine spatial normalizations were performed using SPM8. [¹⁸F]FDG data were quantified using (1) an image-derived-input function obtained from the liver (cMR_glc), using (2) standardized uptake values (SUV_glc) corrected for blood glucose levels and by (3) normalizing counts to the whole-brain uptake. Parametric [¹⁸F]FECT binding images were constructed by reference to the cerebellum. Registration accuracy was determined using random simulated misalignments and vectorial mismatch determination.

Results

Registration accuracy was between 0.21–1.11 mm. Regional intersubject variabilities of cMR_glc ranged from 15.4% to 19.2%, while test-retest values were between 5.0% and 13.0%. For [¹⁸F]FECT uptake in the caudate-putamen, these values were 13.0% and 10.3%, respectively. Regional values of cMR_glc positively correlated to SUV_glc measured within the 45–60 min time frame (spearman r = 0.71). Next, SPM analysis of 6-OHDA-lesioned mice showed hypometabolism in the bilateral caudate-putamen and cerebellum, and an unilateral striatal decrease in DAT availability.

Conclusion

MRM-based small-animal PET templates facilitate accurate assessment and spatial localization of mouse brain function using VOI or voxel-based analysis. Regional intersubject- and test-retest variations indicate that for these targets accuracy comparable to humans can be achieved.     

Development of a Novel PET Tracer [18F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

Background and Objective

The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.

Methods

C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested in vitro and in vivo.

Results

The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.

Conclusions

[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.     

Limits of [18F]-FLT PET as a Biomarker of Proliferation in Oncology

Background

Non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[¹⁸F]-fluorothymidine, [¹⁸F]-FLT. Though several studies have associated serial changes in [¹⁸F]-FLT-PET with elements of therapeutic response, the degree to which [¹⁸F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [¹⁸F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research.

Methods and Findings

[¹⁸F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [¹⁸F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [¹⁸F]-FLT-PET data from standard estimates of proliferative index.

Conclusions

Our findings illustrate that [¹⁸F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [¹⁸F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [¹⁸F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [¹⁸F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [¹⁸F]-FLT-PET correlative results previously reported and suggest future best-practices when [¹⁸F]-FLT-PET is employed in oncology.     

Uptake of [18F] EF5 as a Tracer for Hypoxic and Aggressive Phenotype in Experimental Head and Neck Squamous Cell Carcinoma

PURPOSE: This study aims to investigate whether the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide ([¹⁸F]EF5) and 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) is associated with a hypoxia-driven adverse phenotype in head and neck squamous cell carcinoma cell lines and tumor xenografts. METHODS: Xenografts were imaged in vivo, and tumor sections were stained for hypoxia-inducible factor 1α (Hif-1α), carbonic anhydrase IX (CA IX), and glucose transporter 1 (Glut-1). Tracer uptakes and the expression of Hif-1α were determined in cell lines under 1% hypoxia. RESULTS: High [¹⁸F]EF5 uptake was seen in xenografts expressing high levels of CA IX, Glut-1, and Hif-1α, whereas low [¹⁸F]EF5 uptake was detected in xenografts expressing low amounts of CA IX and Hif-1α. The uptake of [¹⁸F]EF5 between cell lines varied extensively under normoxic conditions. A clear correlation was found between the expression of Hif-1α and the uptake of [¹⁸F]FDG during hypoxia. CONCLUSIONS: The UT-SCC cell lines studied differed with respect to their hypoxic phenotypes, and these variations were detectable with [¹⁸F]EF5. Acute hypoxia increases [¹⁸F]FDG uptake in vitro, whereas a high [¹⁸F]EF5 uptake reflects a more complex phenotype associated with hypoxia and an aggressive growth pattern.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

[18F]DPA-714: Direct Comparison with [11C]PK11195 in a Model of Cerebral Ischemia in Rats

Purpose

Neuroinflammation is involved in several brain disorders and can be monitored through expression of the translocator protein 18 kDa (TSPO) on activated microglia. In recent years, several new PET radioligands for TSPO have been evaluated in disease models. [¹⁸F]DPA-714 is a TSPO radiotracer with great promise; however results vary between different experimental models of neuroinflammation. To further examine the potential of [¹⁸F]DPA-714, it was compared directly to [¹¹C]PK11195 in experimental cerebral ischaemia in rats.

Methods

Under anaesthesia, the middle cerebral artery of adult rats was occluded for 60 min using the filament model. Rats were allowed recovery for 5 to 7 days before one hour dynamic PET scans with [¹¹C]PK11195 and/or [¹⁸F]DPA-714 under anaesthesia.

Results

Uptake of [¹¹C]PK11195 vs [¹⁸F]DPA-714 in the ischemic lesion was similar (core/contralateral ratio: 2.84±0.67 vs 2.28±0.34 respectively), but severity of the brain ischemia and hence ligand uptake in the lesion appeared to vary greatly between animals scanned with [¹¹C]PK11195 or with [¹⁸F]DPA-714. To solve this issue of inter-individual variability, we performed a direct comparison of [¹¹C]PK11195 and [¹⁸F]DPA-714 by scanning the same animals sequentially with both tracers within 24 h. In this direct comparison, the core/contralateral ratio (3.35±1.21 vs 4.66±2.50 for [¹¹C]PK11195 vs [¹⁸F]DPA-714 respectively) showed a significantly better signal-to-noise ratio (1.6 (1.3–1.9, 95%CI) fold by linear regression) for [¹⁸F]DPA-714.

Conclusions

In a clinically relevant model of neuroinflammation, uptake for both radiotracers appeared to be similar at first, but a high variability was observed in our model. Therefore, to truly compare tracers in such models, we performed scans with both tracers in the same animals. By doing so, our result demonstrated that [¹⁸F]DPA-714 displayed a higher signal-to-noise ratio than [¹¹C]PK11195. Our results suggest that, with the longer half-life of [¹⁸F] which facilitates distribution of the tracer across PET centre, [¹⁸F]DPA-714 is a good alternative for TSPO imaging.     

Early Assessment of the Efficacy of Temozolomide Chemotherapy in Experimental Glioblastoma Using [18F]FLT-PET Imaging

Addition of temozolomide (TMZ) to radiation therapy is the standard treatment for patients with glioblastoma (GBM). However, there is uncertainty regarding the effectiveness of TMZ. Considering the rapid evolution of the disease, methods to assess TMZ efficacy early during treatment would be of great benefit. Our aim was to monitor early effects of TMZ in a mouse model of GBM using positron emission tomography (PET) with 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT).

Methods

Human glioma cells sensitive to TMZ (Gli36dEGFR-1) were treated with sub-lethal doses of TMZ to obtain cells with lower sensitivity to TMZ (Gli36dEGFR-2), as measured by growth and clonogenic assays. Gli36dEGFR-1 and Gli36dEGFR-2 cells were subcutaneously (s.c.) or intracranially (i.c.) xenografted into nude mice. Mice were treated for 7 days with daily injection of 25 or 50 mg/kg TMZ. Treatment efficacy was measured using [¹⁸F]FLT-PET before treatment and after 2 days. Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) were used to determine tumor volumes before treatment and after 7 days.

Results

A significant difference was observed between TMZ and DMSO treated tumors in terms of variations of [¹⁸F]FLT T/B ratio as soon as day 2 in the i.c. as well as in the s.c. mouse model. Variations of [¹⁸F]FLT T/B uptake ratio between days 0 and 2 correlated with variations of tumor size between days 0 and 7 (s.c. model: n_tumor = 17 in n_mice = 11, P<0.01; i.c. model: n_tumor/mice = 9, P<0.01).

Conclusions

Our results indicate that [¹⁸F]FLT-PET may be useful for an early evaluation of the response of GBM to TMZ chemotherapy in patients with glioma.     

Early [18F]FET-PET in Gliomas after Surgical Resection: Comparison with MRI and Histopathology

Background

The precise definition of the post-operative resection status in high-grade gliomas (HGG) is crucial for further management. We aimed to assess the feasibility of assessment of the resection status with early post-operative positron emission tomography (PET) using [¹⁸F]O-(2-[¹⁸F]-fluoroethyl)-L-tyrosine ([¹⁸F]FET).

Methods

25 patients with the suspicion of primary HGG were enrolled. All patients underwent pre-operative [¹⁸F]FET-PET and magnetic resonance imaging (MRI). Intra-operatively, resection status was assessed using 5-aminolevulinic acid (5-ALA). Imaging was repeated within 72h after neurosurgery. Post-operative [¹⁸F]FET-PET was compared with MRI, intra-operative assessment and clinical follow-up.

Results

[¹⁸F]FET-PET, MRI and intra-operative assessment consistently revealed complete resection in 12/25 (48%) patients and incomplete resection in 6/25 cases (24%). In 7 patients, PET revealed discordant findings. One patient was re-resected. 3/7 experienced tumor recurrence, 3/7 died shortly after brain surgery.

Conclusion

Early assessment of the resection status in HGG with [¹⁸F]FET-PET seems to be feasible.     

Assessment of radionuclide impurities in [18F]fluoromethylcholine ([18F]FMCH)

Purpose[¹⁸F]Fluoromethylcholine ([¹⁸F]FMCH) is a radiopharmaceutical used in positron emission tomography (PET) imaging for the study of prostate, breast, and brain tumors. It is usually synthesized in cyclotron facilities where ¹⁸F is produced by proton irradiation of [¹⁸O]H₂O through ¹⁸O(p,n)¹⁸F reaction. Due to the activation of target materials, the bombardment causes unwanted radionuclidic impurities in [¹⁸O]H₂O, that need to be removed during the radiopharmaceutical synthesis. Thus, the aim of this study is to quantify the radionuclide impurities in the ¹⁸F production process and in the synthesized [¹⁸F]FMCH, demonstrating the radionuclidic purity of this radiopharmaceutical.MethodsLong-lived radionuclide impurities were experimentally assessed using high-resolution gamma and liquid scintillation spectrometries, while short-lived impurities were monitored analyzing the decay curve of the irradiated [¹⁸O]H₂O with an activity calibrator. As spectrometric radionuclide library, a Geant4 Monte Carlo simulation of the ¹⁸F-target assembly was previously performed.Results³H, ^52,54Mn, ^56,57,58Co, ^95m,96Tc, ¹⁰⁹Cd, and ¹⁸⁴Re were found in the irradiated [¹⁸O]H₂O, but no radionuclide was found in the non-irradiated [¹⁸O]H₂O neither in the final [¹⁸F]FMCH solution with an activity concentration greater than the minimum detectable activity concentration. A total impurity activity <6.2 kBq was measured in the irradiated [¹⁸O]H₂O, whereas a [¹⁸F]FMCH radionuclide purity >99.9999998% was estimated. Finally, the decay curve of the irradiated [¹⁸O]H₂O revealed a very low maximum of ¹³N activity (<0.03% of ¹⁸F) even immediately after the end of bombardment.ConclusionsThis study demonstrated the radionuclidic purity of [¹⁸F]FMCH according to the EU Pharmacopeia.     

An Intra-Individual Comparison of MRI, [18F]-FET and [18F]-FLT PET in Patients with High-Grade Gliomas

Objectives

Intra-individual spatial overlap analysis of tumor volumes assessed by MRI, the amino acid PET tracer [¹⁸F]-FET and the nucleoside PET tracer [¹⁸F]-FLT in high-grade gliomas (HGG).

Methods

MRI, [¹⁸F]-FET and [¹⁸F]-FLT PET data sets were retrospectively analyzed in 23 HGG patients. Morphologic tumor volumes on MRI (post-contrast T1 (cT1) and T2 images) were calculated using a semi-automatic image segmentation method. Metabolic tumor volumes for [¹⁸F]-FET and [¹⁸F]-FLT PETs were determined by image segmentation using a threshold-based volume of interest analysis. After co-registration with MRI the morphologic and metabolic tumor volumes were compared on an intra-individual basis in order to estimate spatial overlaps using the Spearman''s rank correlation coefficient and the Mann-Whitney U test.

Results

[¹⁸F]-FLT uptake was negative in tumors with no or only moderate contrast enhancement on MRI, detecting only 21 of 23 (91%) HGG. In addition, [¹⁸F]-FLT uptake was mainly restricted to cT1 tumor areas on MRI and [¹⁸F]-FLT volumes strongly correlated with cT1 volumes (r = 0.841, p<0.001). In contrast, [¹⁸F]-FET PET detected 22 of 23 (96%) HGG. [¹⁸F]-FET uptake beyond areas of cT1 was found in 61% of cases and [¹⁸F]-FET volumes showed only a moderate correlation with cT1 volumes (r = 0.573, p<0.001). Metabolic tumor volumes beyond cT1 tumor areas were significantly larger for [¹⁸F]-FET compared to [¹⁸F]-FLT tracer uptake (8.3 vs. 2.7 cm³, p<0.001).

Conclusion

In HGG [¹⁸F]-FET but not [¹⁸F]-FLT PET was able to detect metabolic active tumor tissue beyond contrast enhancing tumor on MRI. In contrast to [¹⁸F]-FET, blood-brain barrier breakdown seems to be a prerequisite for [¹⁸F]-FLT tracer uptake.     

Early detection of response to experimental chemotherapeutic Top216 with [18F]FLT and [18F]FDG PET in human ovary cancer xenografts in mice

Background

3′-deoxy-3′-[¹⁸F]fluorothymidine (¹⁸F-FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to use ¹⁸F-FLT positron emission tomography (PET) to study treatment responses to a new anti-cancer compound. To do so, we studied early anti-proliferative effects of the experimental chemotherapy Top216 non-invasively by PET.

Methodology/Principal Findings

In vivo uptake of ¹⁸F-FLT in human ovary cancer xenografts in mice (A2780) was studied at various time points after Top216 treatment (50 mg/kg i.v. at 0 and 48 hours) was initiated. Baseline ¹⁸F-FLT scans were made before either Top216 (n = 7–10) or vehicle (n = 5–7) was injected and repeated after 2 and 6 hours and 1 and 5 days of treatment. A parallel study was made with 2′-deoxy-2′-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) (n = 8). Tracer uptake was quantified using small animal PET/CT. Imaging results were validated by tumor volume changes and gene-expression of Ki67 and TK1. Top216 (50 mg/kg 0 and 48 hours) inhibited the growth of the A2780 tumor compared to the control group (P<0.001). ¹⁸F-FLT uptake decreased significantly at 2 hours (−52%; P<0.001), 6 hours (−49%; P = 0.002) and Day 1 (−47%; P<0.001) after Top216 treatment. At Day 5 ¹⁸F-FLT uptake was comparable to uptake in the control group. Uptake of ¹⁸F-FLT was unchanged in the control group during the experiment. In the treatment group, uptake of ¹⁸F-FDG was significantly decreased at 6 hours (−21%; P = 0.003), Day 1 (−29%; P<0.001) and Day 5 (−19%; P = 0.05) compared to baseline.

Conclusions/Significance

One injection with Top216 initiated a fast and significant decrease in cell-proliferation assessable by ¹⁸F-FLT after 2 hours. The early reductions in tumor cell proliferation preceded changes in tumor size. Our data indicate that ¹⁸F-FLT PET is promising for the early non-invasive assessment of chemotherapy effects in both drug development and for tailoring therapy in patients.     

Synthesis,radiofluorination, and hypoxia-selective studies of FRAZ: A configurational and positional analogue of the clinical hypoxia marker, [18F]-FAZA

The current work evaluates 1-α-d-(2-deoxy-2-fluororibofuranosyl)-2-nitroimidazole (FRAZ), a novel azomycin nucleoside that is a potential radiosensitizer of tumor hypoxia. FRAZ is a ribose analogue of 1-α-d-(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole ([¹⁸F]-FAZA), a clinically used hypoxia marker. Preliminary assessment of the cytotoxicity and hypoxia-specific in vitro binding in HCT-110 colorectal cancer cells indicate that the radiosensitization properties of FRAZ are similar to that of FAZA, with a sensitizer enhancement ratio (SER) of ～1.8. An automated radiosynthesis of [¹⁸F]-FRAZ using a commercial automated synthesis unit (ASU) was established (synthesis time ～32 min; radiochemical yield (decay uncorrecetd) ～22%) to facilitate its application in PET-based diagnosis of hypoxic tumors.     

Evaluation of the novel folate receptor ligand [18F]fluoro-PEG-folate for macrophage targeting in a rat model of arthritis

Introduction

Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[¹¹C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [¹⁸F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.

Methods

[¹⁸F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [¹⁸F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[¹¹C]PK11195.

Results

[¹⁸F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [¹⁸F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [¹⁸F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [¹⁸F]fluoro-PEG-folate were increased compared with those of (R)-[¹¹C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [¹⁸F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.

Conclusions

The novel PET tracer [¹⁸F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[¹¹C]PK11195. These results warrant further exploration of [¹⁸F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.     

[18F]FEAC and [18F]FEDAC: Two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain

[¹⁸F]FEAC ([¹⁸F]4a) and [¹⁸F]FEDAC ([¹⁸F]4b) were developed as two novel positron emission tomography (PET) ligands for peripheral-type benzodiazepine receptor (PBR). [¹⁸F]4a and [¹⁸F]4b were synthesized by fluoroethylation of precursors 8a and 8b with [¹⁸F]FCH₂CH₂Br ([¹⁸F]9), respectively. Small-animal PET scan for a neuroinflammatory rat model showed that the two radioligands had high uptakes of radioactivity in the kainic acid-infused striatum, a brain region where PBR density was increased.     

Synthesis and bioevaluation of [18F]4-fluoro-m-hydroxyphenethylguanidine ([18F]4F-MHPG): A novel radiotracer for quantitative PET studies of cardiac sympathetic innervation

A new cardiac sympathetic nerve imaging agent, [¹⁸F]4-fluoro-m-hydroxyphenethylguanidine ([¹⁸F]4F-MHPG), was synthesized and evaluated. The radiosynthetic intermediate [¹⁸F]4-fluoro-m-tyramine ([¹⁸F]4F-MTA) was prepared and then sequentially reacted with cyanogen bromide and NH₄Br/NH₄OH to afford [¹⁸F]4F-MHPG. Initial bioevaluations of [¹⁸F]4F-MHPG (biodistribution studies in rats and kinetic studies in the isolated rat heart) were similar to results previously reported for the carbon-11 labeled analog [¹¹C]4F-MHPG. The neuronal uptake rate of [¹⁸F]4F-MHPG into the isolated rat heart was 0.68 ml/min/g wet and its retention time in sympathetic neurons was very long (T_1/2 >13 h). A PET imaging study in a nonhuman primate with [¹⁸F]4F-MHPG provided high quality images of the heart, with heart-to-blood ratios at 80–90 min after injection of 5-to-1. These initial kinetic and imaging studies of [¹⁸F]4F-MHPG suggest that this radiotracer may allow for more accurate quantification of regional cardiac sympathetic nerve density than is currently possible with existing neuronal imaging agents.     

PET Imaging of Lung Inflammation with [18F]FEDAC, a Radioligand for Translocator Protein (18 kDa)

Purpose

The translocator protein (18 kDa) (TSPO) is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lung. This study aimed to perform positron emission tomography (PET) imaging of lung inflammation with [¹⁸F]FEDAC, a specific TSPO radioligand, and to determine cellular sources enriching TSPO expression in the lung.

Methods

An acute lung injury model was prepared by intratracheal administration of lipopolysaccharide (LPS) to rat. Uptake of radioactivity in the rat lungs was measured with small-animal PET after injection of [¹⁸F]FEDAC. Presence of TSPO was examined in the lung tissue using Western blot and immunohistochemical assays.

Results

The uptake of [¹⁸F]FEDAC increased in the lung with the progress of inflammation by treatment with LPS. Pretreatment with a TSPO-selective ligand PK11195 showed a significant decrease in the lung uptake of [¹⁸F]FEDAC due to competitive binding to TSPO. TSPO expression was elevated in the inflamed lung section and its level responded to the [¹⁸F]FEDAC uptake and severity of inflammation. Increase of TSPO expression was mainly found in the neutrophils and macrophages of inflamed lungs.

Conclusion

From this study we conclude that PET with [¹⁸F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation. Study on human lung using [¹⁸F]FEDAC-PET is promising.     

Preparation and biodistribution of [18F]FP2OP as myocardial perfusion imaging agent for positron emission tomography

Myocardial extractions of pyridaben, a mitochondrial complex I (MC-I) inhibitor, is well correlated with blood flow. Based on the synthesis and characterization of pyridaben analogue 2-tert-butyl-5-[2-(2-[¹⁸F]fluroethoxy)ethoxy]benzyloxy]-4-chloro-2H-pyridazin-3-one ([¹⁸F]FP2OP), this study assessed its potential to be developed as myocardial perfusion imaging (MPI) agent.Methods: The tosylate labeling precursor 2-(2-(4-(tert-butyl-5-chloro-6-oxo-1,6-dihydro-pyridazin-4-yloxymethyl)benzyloxy)ethoxy)ethyl ester (OTs-P2OP) and the nonradioactive 2-tert-butyl-5-[2-(2-[¹⁹F]fluroethoxy)ethoxy]benzyloxy]-4-chloro-2H-pyridazin-3-one ([¹⁹F]FP2OP) were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and MS analysis. By substituting tosyl of precursor OTs-P2OP with ¹⁸F, the radiolabeled complex [¹⁸F]FP2OP was prepared and further evaluated for its in vitro physicochemical properties, in vivo biodistribution, the metabolic stability in mice, ex vivo autoradiography and cardiac PET/CT imaging.Results: Starting with [¹⁸F]F^? Kryptofix 2.2.2./K₂CO₃ solution, the total reaction time for [¹⁸F]FP2OP was about 100 min, with final high-performance liquid chromatography purification included. Typical decay-corrected radiochemical yield stayed at 41 ± 5.3%, the radiochemical purity, 98% or more. Biodistribution in mice showed that the heart uptake of [¹⁸F]FP2OP was 41.90 ± 4.52%ID/g at 2 min post-injection time, when the ratio of heart/liver, heart/lung and heart/blood reached 6.83, 9.49 and 35.74, respectively. Lipophilic molecule was further produced by metabolized [¹⁸F]FP2OP in blood and urine at 30 min. Ex vivo autoradiography demonstrates that [¹⁸F]FP2OP may have high affinity with MC-I and that can be blocked by [¹⁹F]FP2OP or rotenone (a known MC-I inhibitor). Cardiac PET images were obtained in a Chinese mini-swine at 5, 15, 30 and 60 min post-injection time with high quality.Conclusion: [¹⁸F]FP2OP was synthesized with high radiochemical yield. The promising biological properties of [¹⁸F]FP2OP suggest high potential as MPI agent for positron emission tomography in the future.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.     

Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with ¹⁸F (t_½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for ¹⁸F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [¹⁸F]AlF-NOTA moiety with [¹⁸F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[¹⁸F]fluoronicotinyl-PEG₄-methyltetrazine to provide [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB) also were synthesized. Radiochemical purity, affinity (K_D) and immunoreactive fraction of [¹⁸F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [¹⁸F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[¹²⁵I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [¹⁸F]FN-GK-5F7 were 3–4 times higher than those for co-injected iso-[¹²⁵I]SGMIB-5F7 as well as those observed for the ¹⁸F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [¹⁸F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [¹⁸F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.