A Novel <Emphasis Type="Italic">In Vitro</Emphasis> Assay to Assess Phosphorylation of 3′-[<Superscript>18</Superscript>F]fluoro-3′-Deoxythymidine

Purpose  

3′-[¹⁸F]fluoro-3'-deoxythymidine ([¹⁸F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [¹⁸F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [¹⁸F]FLT both in tumor cell lysates and tumor cells.     

Reduction of [<Superscript>18</Superscript>F]Fluoromisonidazole Uptake After Neoadjuvant Chemotherapy for Head and Neck Squamous Cell Carcinoma

Purpose  

The purpose of this study was to investigate the changes of tumor hypoxia as a result of neoadjuvant chemotherapy (NAC) by measuring the changes of [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) positron emission tomography (PET) uptake, as well as to look into the ability of [¹⁸F]FMISO PET to predict the NAC result.     

Tracer Level Electrophilic Synthesis and Pharmacokinetics of the Hypoxia Tracer [18F]EF5

Purpose  

2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide labeled with [¹⁸F]-fluorine ([¹⁸F]EF5), a promising tracer for tumor hypoxia, has previously been synthesized in low yields and low specific radioactivity. In pharmacokinetic evaluations, in the presence of non-radioactive EF5, a uniform and low background uptake and high in vivo stability of [¹⁸F]EF5 have been demonstrated. Our purpose was to increase the specific radioactivity of [¹⁸F]EF5 to enable to study the pharmacokinetics at trace level.     

Preclinical Imaging of Therapy Response Using Metabolic and Apoptosis Molecular Imaging

Purpose  

Early after therapy, 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) imaging is not always reliable due to the influx of inflammatory cells while apoptosis imaging offers a direct and early measurement of therapy effects. This study uses an improved apoptosis probe (^99mTc-hAnxA5) in combination with [¹⁸F]FDG imaging to evaluate therapy response.     

Electrophilic Addition of Chlorine Monofluoride for PET tracers

Purpose

We have studied the utility of [¹⁸F]ClF electrophilic addition to the carbon–carbon double bond of analogues of a model positron emission tomography (PET) tracer, [¹⁸F]EF5. The consequence of simultaneous chlorine/fluorine addition on lipophilicity and biological activity of the molecule is evaluated.

Procedures

Post-target produced [¹⁸F]F₂ was reacted with Cl₂ to produce [¹⁸F]ClF, which was used in electrophilic addition.

Results

[¹⁸F]ClF was produced and used to label chlorinated analogues of [¹⁸F]EF5. The chlorinated analogues, [¹⁸F]EF4Cl_a and [¹⁸F]EF4Cl_b, were synthesized simultaneously. The in vivo uptake of the analogues compared well with [¹⁸F]EF5 uptake in tumor-bearing mice.

Conclusion

[¹⁸F]ClF is a suitable labeling reagent for electrophilic addition to double bonds of PET tracers. The results show that the modification of the pentafluoro group of [¹⁸F]EF5 by monofluorine-for-chlorine exchange affected the lipophilicity, but the hypoxia avidity of these molecules was not apparently altered.     

<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Radiofluorinated Caspase-3/7 Inhibitors as Radiotracers for Apoptosis Imaging and Comparison with [<Superscript>18</Superscript>F]ML-10 in a Stroke Model in the Rat

Purpose

The first biological evaluation of two potent fluorine-18 radiolabelled inhibitors of caspase-3/7 was achieved in a cerebral stroke rat model to visualize apoptosis.

Procedures

In vivo characteristics of isatins [¹⁸F]-2 and [¹⁸F]-3 were studied and compared by μPET to previously described 1-[4-(2-[¹⁸F]fluoroethyl)benzyl]-5-(2-methoxymethylpyrrolidin-1-ylsulfonyl)isatin ([¹⁸F]-1) and to 2-(5-[¹⁸F]fluoropentyl)-2-methyl-malonic acid ([¹⁸F]ML-10) used as a reference radiotracer in a rat stroke model.

Results

[¹⁸F]-2 and [¹⁸F]-3 were radiolabelled with high radiochemical purity and high specific radioactivity. Radioactivity uptakes in ischemic and contralateral brain regions were weak for the three radiolabelled isatins and lower for [¹⁸F]ML-10. In μPET, time activity curves showed significant uptake differences between both regions of interest for [¹⁸F]-1 after 45 min. No differences were observed for [¹⁸F]ML-10.

Conclusions

Radiolabelled isatins are more promising radiotracers to image apoptosis than [¹⁸F]ML-10 in this stroke animal model without craniectomy. In particular, [¹⁸F]-1 presented significant uptake in apoptotic area 45 min after administration

     

Simultaneous Dual-tracer PET Imaging of the Rat Brain and its Application in the Study of Cerebral Ischemia

Purpose  

This study evaluates the performance of simultaneous dual-tracer technique (SDTT) in static positron emission tomography (PET) studies using 2-deoxy-2-[¹⁸F]fluoro-D-glucose and [¹³N]ammonium as radiotracers.     

Comparison of 2β-Carbomethoxy-3β-(4-[<Superscript>18</Superscript>F]Fluorophenyl)Tropane and <Emphasis Type="Italic">N</Emphasis>-(3-[<Superscript>18</Superscript>F]Fluoropropyl)-2β-Carbomethoxy-3β-(4-Fluorophenyl)Nortropane,Tracers for Imaging Dopamine Transporter in Rat

Purpose

This study compares 2β-carbomethoxy-3β-(4-[¹⁸F]fluorophenyl)tropane ([¹⁸F]β-CFT) and N-(3-[¹⁸F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([¹⁸F]β-CFT-FP) as radiotracers for imaging the dopamine transporter (DAT) in rat.

Procedures

Biodistribution, specificity and selectivity of the radiotracers were studied ex vivo in rats pre-treated with specific antagonists for DAT, serotonin transporter (SERT) and noradrenalin transporter (NET) and in control rats. Positron emission tomography (PET) studies were performed using an HRRT scanner. Radiolabelled metabolites were analyzed with thin-layer chromatography.

Results

[¹⁸F]β-CFT showed slow kinetics with a maximum striatum/cerebellum uptake ratio of 9.2 at 120 min. [¹⁸F]β-CFT-FP showed fast kinetics with a maximum ratio of 3.1 at 5 min. Both tracers bound to DAT. [¹⁸F]β-CFT also bound to NET. [¹⁸F]β-CFT was more resistant to metabolism than [¹⁸F]β-CFT-FP.

Conclusions

Structural modifications of [¹⁸F]β-CFT significantly changed its biological properties, as shown by [¹⁸F]β-CFT-FP. [¹⁸F]β-CFT is a suitable tracer for both preclinical and human PET studies, but [¹⁸F]β-CFT-FP is less suitable as a PET tracer.

     

Fully Automated Preparation and Conjugation of <Emphasis Type="Italic">N</Emphasis>-Succinimidyl 4-[<Superscript>18</Superscript>F]Fluorobenzoate ([<Superscript>18</Superscript>F]SFB) with RGD Peptide Using a GE FASTlab™ Synthesizer

Purpose  

The aim of this work was to automate the radiosynthesis of [¹⁸F]SFB, a widely used reagent for the labeling of biomolecules with ¹⁸F on a new generation commercial synthesis module (FASTLab™, GE Healthcare).     

In Vitro and In Vivo Evaluation of the Caspase-3 Substrate-Based Radiotracer [18F]-CP18 for PET Imaging of Apoptosis in Tumors

Purpose

A novel caspase-3 substrate-based probe [¹⁸F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors.

Methods

Uptake of [¹⁸F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis.

Results

The in vitro cell assays showed caspase-3-dependent uptake of [¹⁸F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [¹⁸F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [¹⁸F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC).

Conclusions

[¹⁸F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [¹⁸F]-CP18 as a promising new PET imaging agent for apoptosis.     

Preclinical Imaging Evaluation of Novel TSPO-PET Ligand 2-(5,7-Diethyl-2-(4-(2-[18F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ([18F]VUIIS1008) in Glioma

Purpose

Translocator protein (TSPO) concentrations are elevated in glioma, suggesting a role for TSPO positron emission tomography (PET) imaging in this setting. In preclinical PET studies, we evaluated a novel, high-affinity TSPO PET ligand, [¹⁸F]VUIIS1008, in healthy mice and glioma-bearing rats.

Procedures

Dynamic PET data were acquired simultaneously with [¹⁸F]VUIIS1008 injection, with binding reversibility and specificity evaluated in vivo by non-radioactive ligand displacement or blocking. Compartmental analysis of PET data was performed using metabolite-corrected arterial input functions. Imaging was validated with histology and immunohistochemistry.

Results

[¹⁸F]VUIIS1008 exhibited rapid uptake in TSPO-rich organs. PET ligand uptake was displaceable with non-radioactive VUIIS1008 or PBR06 in mice. Tumor accumulation of [¹⁸F]VUIIS1008 was blocked by pretreatment with VUIIS1008 in rats. [¹⁸F]VUIIS1008 exhibited improved tumor-to-background ratio and higher binding potential in tumors compared to a structurally similar pyrazolopyrimidine TSPO ligand, [¹⁸F]DPA-714.

Conclusions

The PET ligand [¹⁸F]VUIIS1008 exhibits promising characteristics as a tracer for imaging glioma. Further translational studies appear warranted.     

Evaluation of [18F]-CP18 as a PET Imaging Tracer for Apoptosis

Purpose

We identified and validated [¹⁸F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells.

Procedures

CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [¹⁸F]-CP18 were assessed in vivo. [¹⁸F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity.

Results

In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [¹⁸F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [¹⁸F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice.

Conclusions

We report the use [¹⁸F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.     

Increase of [18F]FLT Tumor Uptake In Vivo Mediated by FdUrd: Toward Improving Cell Proliferation Positron Emission Tomography

Purpose

3??-deoxy-3??-[¹⁸F]fluorothymidine ([¹⁸F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2??-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [¹⁸F]FLT in tumor xenografts.

Methods

The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[¹²⁵I]iodo-2??-deoxyuridine biodistribution measurements. In [¹⁸F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [¹⁸F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [¹⁸F]FDG tumor uptake. Using microPET, the dynamic distribution of [¹⁸F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation.

Results

FdUrd induced an immediate increase in tumor uptake of 5-[¹²⁵I]iodo-2??-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [¹⁸F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [¹⁸F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [¹⁸F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [¹⁸F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis.

Conclusion

We showed a reliable and significant uptake increase of [¹⁸F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [¹⁸F]FLT PET.     

Shortening the Duration of [<Superscript>18</Superscript>F]FDG PET Brain Examination for Diagnosis of Brain Glioma

Purpose  

Some patients cannot remain immobile for a long duration of 60 min, which is generally applied in the case of a 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) dynamic positron emission tomography (PET) scan. We investigated the change of the parametric values when the time duration of PET data was shortened.     

Fully Automated Radiosynthesis of 2-[18F]Fludarabine for PET Imaging of Low-Grade Lymphoma

Purpose

An efficient and fully automated radiosynthesis of 2-[¹⁸F]fluoro-9-β-d-arabinofuranosyl-adenine (2-[¹⁸F]fludarabine, [¹⁸F]-5) based on a GE TRACERlab? FX-FN module has been developed.

Procedures

A 2-nitro purine derivative 3 was developed as precursor for labeling with fluorine-18. The radiosynthesis of [¹⁸F]-5 was performed in two steps in a single reactor with an intermediary purification on Sep-Pak® silica which involved the addition of a three-way valve on the original module. After hydrolysis, [¹⁸F]-5 was purified by semi-preparative high-pressure liquid chromatography (HPLC) and a quality control was established.

Results

The labeling precursor 3 was obtained in 45 % overall yield. Nucleophilic substitution with K¹⁸F/K_2.2.2 afforded protected 2-[¹⁸F]fludarabine ([¹⁸F]-4) in 73?±?4 % , radiochemical yield (decay corrected to the end of bombardment (EOB)) and based on the initial [¹⁸F]F^? activity. An aqueous ammonia/methanol solution was used for the deprotection reaction and gave the desired [¹⁸F]-5 in 67?±?3 % yield after 20 min at 70 °C based on HPLC profile.

Conclusions

The process afforded pure 2-[¹⁸F]fludarabine in 48?±?3 % yield (decay corrected to the EOB) in 85 min, with a specific activity of 310?±?72 GBq/μmol at the end of synthesis (EOS) and a radiochemical purity up to 99 %.     

Evaluation of (4-[<Superscript>18</Superscript>F]Fluorophenyl)triphenylphosphonium Ion. A Potential Myocardial Blood Flow Agent for PET

Purpose  

The lipophilic cationic compound, (4-[¹⁸F]fluorophenyl)triphenylphosphonium ion (¹⁸F-FTPP) was synthesized and evaluated as a potential positron emission tomography (PET) myocardial perfusion agent.     

Evaluation of 2-Deoxy-2-[18F]Fluoro-D-glucose- and 3′-Deoxy-3′-[18F]Fluorothymidine–Positron Emission Tomography as Biomarkers of Therapy Response in Platinum-Resistant Ovarian Cancer

Purpose

We evaluated whether 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and 3??-deoxy-3??-[¹⁸F]fluorothymidine ([¹⁸F]FLT) positron emission tomography (PET) could be used as imaging biomarkers of platinum resensitization in ovarian cancer.

Procedures

Paired platinum-sensitive and platinum-resistant ovarian cancer cells from the same patient, PEO1 and PEO4, grown as tumor xenografts in nude mice, were assessed by PET.

Results

The AKT inhibitor, API-2, resensitized platinum-resistant PEO4 tumors to cisplatin, leading to a markedly lower Ki67 labeling index (p????0.006, n?=?6 per group). [¹⁸F]FDG-PET and [¹⁸F]FLT-PET imaging variables were lower after combination treatment compared with vehicle treatment (p????0.006, n?=?6 per group). No changes were seen with either drug alone. PRAS40 phosphorylation status was a sensitive biochemical marker of pathway inhibition, whereas reductions thymidine kinase 1 expression defined the [¹⁸F]FLT response.

Conclusions

Therapeutic inhibition of AKT activation in acquired platinum-resistant disease can be imaged noninvasively by [¹⁸F]FDG-PET and [¹⁸F]FLT-PET warranting further assessment.     

Feasibility of Assessing [18F]FDG Lung Metabolism with Late Dynamic PET Imaging

Purpose  

The aim of this work was to non-invasively establish the feasibility of assessing 2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) lung metabolism with the use of a late dynamic positron emission tomograpy (PET) acquisition, i.e., beyond 2 h after injection.     

An Evaluation of 2-deoxy-2-[18F]Fluoro-D-Glucose and 3′-deoxy-3′-[18F]-Fluorothymidine Uptake in Human Tumor Xenograft Models

Purpose

The aim of this study is to assess the variability of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]-FDG) and 3??-deoxy-3??-[¹⁸F]-fluorothymidine ([¹⁸F]-FLT) uptake in pre-clinical tumor models and examine the relationship between imaging data and related histological biomarkers.

Procedures

[¹⁸F]-FDG and [¹⁸F]-FLT studies were carried out in nine human tumor xenograft models in mice. A selection of the models underwent histological analysis for endpoints relevant to radiotracer uptake. Comparisons were made between in vitro uptake, in vivo imaging, and ex vivo histopathology data using quantitative and semi-quantitative analysis.

Results

In vitro data revealed that [1-¹⁴C]-2-deoxy-d-glucose ([¹⁴C]-2DG) uptake in the tumor cell lines was variable. In vivo, [¹⁸F]-FDG and [¹⁸F]-FLT uptake was highly variable across tumor types and uptake of one tracer was not predictive for the other. [¹⁴C]-2DG uptake in vitro did not predict for [¹⁸F]-FDG uptake in vivo. [¹⁸F]-FDG SUV was inversely proportional to Ki67 and necrosis levels and positively correlated with HKI. [¹⁸F]-FLT uptake positively correlated with Ki67 and TK1.

Conclusion

When evaluating imaging biomarkers in response to therapy, the choice of tumor model should take into account in vivo baseline radiotracer uptake, which can vary significantly between models.     

Evaluation of TSPO PET Ligands [<Superscript>18</Superscript>F]VUIIS1009A and [<Superscript>18</Superscript>F]VUIIS1009B: Tracers for Cancer Imaging

Purpose

Positron emission tomography (PET) ligands targeting translocator protein (TSPO) are potential imaging diagnostics of cancer. In this study, we report two novel, high-affinity TSPO PET ligands that are 5,7 regioisomers, [¹⁸F]VUIIS1009A ([¹⁸F]3A) and [¹⁸F]VUIIS1009B ([¹⁸F]3B), and their initial in vitro and in vivo evaluation in healthy mice and glioma-bearing rats.

Procedures

VUIIS1009A/B was synthesized and confirmed by X-ray crystallography. Interactions between TSPO binding pocket and novel ligands were evaluated and compared with contemporary TSPO ligands using 2D ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectroscopy. In vivo biodistribution of [¹⁸F]VUIIS1009A and [¹⁸F]VUIIS1009B was carried out in healthy mice with and without radioligand displacement. Dynamic PET imaging data were acquired simultaneously with [¹⁸F]VUIIS1009A/B injections in glioma-bearing rats, with binding reversibility and specificity evaluated by radioligand displacement. In vivo radiometabolite analysis was performed using radio-TLC, and quantitative analysis of PET data was performed using metabolite-corrected arterial input functions. Imaging was validated with histology and immunohistochemistry.

Results

Both VUIIS1009A (3A) and VUIIS1009B (3B) were found to exhibit exceptional binding affinity to TSPO, with observed IC₅₀ values against PK11195 approximately 500-fold lower than DPA-714. However, HSQC NMR suggested that VUIIS1009A and VUIIS1009B share a common binding pocket within mammalian TSPO (mTSPO) as DPA-714 and to a lesser extent, PK11195. [¹⁸F]VUIIS1009A ([¹⁸F]3A) and [¹⁸F]VUIIS1009B ([¹⁸F]3B) exhibited similar biodistribution in healthy mice. In rats bearing C6 gliomas, both [¹⁸F]VUIIS1009A and [¹⁸F]VUIIS1009B exhibited greater binding potential (k ₃/k ₄)in tumor tissue compared to [¹⁸F]DPA-714. Interestingly, [¹⁸F]VUIIS1009B exhibited significantly greater tumor uptake (V _T) than [¹⁸F]VUIIS1009A, which was attributed primarily to greater plasma-to-tumor extraction efficiency.

Conclusions

The novel PET ligand [¹⁸F]VUIIS1009B exhibits promising characteristics for imaging glioma; its superiority over [¹⁸F]VUIIS1009A, a regioisomer, appears to be primarily due to improved plasma extraction efficiency. Continued evaluation of [¹⁸F]VUIIS1009B as a high-affinity TSPO PET ligand for precision medicine appears warranted.