Simplified fast and high yielding automated synthesis of [18F]fluoroethylcholine for prostate cancer imaging

(11)C- and (18)F-labeled choline analogues are successful tracers for prostate cancer imaging using positron emission tomography (PET). Due to the practical advantages of the longer-living radioisotope (18)F (t(1/2)=110 min) instead of (11)C (t(1/2)=20 min), [(18)F]fluoroethylcholine has been introduced to increase the opportunity of widespread clinical application. Nevertheless, the various known synthetic methods provide [(18)F]fluoroethylcholine for human use only in moderate overall yields of up to 30% so far. Here, a new simplified and high yield two-step-synthesis for [(18)F]fluoroethylcholine is described for potential clinical applications starting from 2-bromoethyl triflate (BETfO) using a modified, commercially available fully automated synthesis module. All synthesis parameters were subsequently optimized resulting in a total yield of 47+/-5% (not decay corrected) in only 40min. [(18)F]fluoroethylcholine could be obtained ready for human use as physiological solution after fixation on Sep-Pak Accell Light cartridges (waters((R))) and formulation with saline without the need of GC or HPLC purification. Radiochemical purity was >99.9% and no contamination of the sterile solution with chemicals used during the synthesis was detected.     

New approach for the synthesis of [F]fluoroethyltyrosine for cancer imaging: Simple, fast, and high yielding automated synthesis

O-(2-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) is one of the first ¹⁸F-labeled amino acids for imaging amino acid metabolism in tumors. This tracer overcomes the disadvantages of [¹⁸F]fluorodeoxyglucose, [¹⁸F]FDG, and [¹¹C]methionine, [¹¹C]MET. Nevertheless, the various synthetic methods providing ¹⁸F[FET] exhibit a big disadvantage concerning the necessity of two purification steps during the synthesis including HPLC purification, which causes difficulties in the automation, moderate yields, and long synthesis times >60 min.A new approach for the synthesis of [¹⁸F]FET is developed starting from 2-bromoethyl triflate as precursor. After optimization of the synthesis parameters including the distillation step of [¹⁸F]-FCH₂CH₂Br combined with the final purification of [¹⁸F]FET using a simple solid phase extraction instead of an HPLC run the synthesis [¹⁸F]FET could be significantly simplified, shortened, and improved. The radiochemical yield (RCY) was about 45% (not decay corrected and calculated relative to [¹⁸F]F⁻ activity that was delivered from the cyclotron). Synthesis time was only 35 min from the end of bombardment (EOB) and the radiochemical purity was >99% at the end of synthesis (EOS). Thus, this simplified synthesis for [¹⁸F]FET offers a very good option for routine clinical use.     

Synthesis of 4-(5-[18F]fluoromethyl-3-phenylisoxazol-4-yl)benzenesulfonamide, a new [18F]fluorinated analogue of valdecoxib, as a potential radiotracer for imaging cyclooxygenase-2 with positron emission tomography

Fluoroalkyl and fluoroaryl analogues of valdecoxib were found to possess potent inhibitory activities against cyclooxygenase-2 comparable to that of the parent valdecoxib. Among them, the fluoromethyl analogue was chosen for 18F-labeling. Thus, 4-(5-[18F]fluoromethyl-3-phenylisoxazol-4-yl)benzenesulfonamide (approximately 2000 Ci/mmol at end of synthesis) was synthesized by [18F]fluoride-ion displacement of the corresponding tosylate in approximately 40% decay-corrected radiochemical yield within approximately 120 min from end of bombardment.     

Synthesis and evaluation of 18F labeled crizotinib derivative [18F]FPC as a novel PET probe for imaging c-MET-positive NSCLC tumor

c-MET-positive NSCLC is an important subtype accounting for about 5%~22% of lung cancer. NSCLC patients with activating c-MET are intensively sensitive to c-MET selective receptor tyrosine kinase (RTK) inhibitors, so we aimed to develop a specific PET probe targeting to c-MET-positive NSCLC for potential patients screened by PET/CT. Herein, PET tracer ¹⁸F-radiolabeled crizotinib derivative ([¹⁸F]FPC) was successfully achieved through a simple one-step ¹⁸F-labeling method. [¹⁸F]FPC PET imaging on c-MET-positive (as well as blocking group) and negative NSCLC models were further evaluated, and results showed that [¹⁸F]FPC was effective as a PET imaging probe that targeted c-MET-positive tumor. Therefore, [¹⁸F]FPC could be a potential PET imaging probe for NSCLC tumor which was sensitive to c-MET-TKIs. By virtue of this property, it will benefit NSCLC patients for c-MET-TKI treatment.     

Prostate cancer PET bioprobes: synthesis of [18F]-radiolabeled hydroxyflutamide derivatives

Approximately 80-90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-propanamide [18F]-1 and N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-2, derivatives of hydroxyflutamide, were synthesized as a fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were successfully labeled with a 10+/-3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of 1500+/-200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molecular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of prostate cancer.     

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT)

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.     

Total synthesis and evaluation of [18F]MHMZ

Radiochemical labeling of MDL 105725 using the secondary labeling precursor 2-[(18)F]fluoroethyltosylate ([(18)F]FETos) was carried out in yields of approximately 90% synthesizing [(18)F]MHMZ in a specific activity of approximately 50MBq/nmol with a starting activity of approximately 3GBq. Overall radiochemical yield including [(18)F]FETos synthon synthesis, [(18)F]fluoroalkylation and preparing the injectable [(18)F]MHMZ solution was 42% within a synthesis time of approximately 100 min. The novel compound showed excellent specific binding to the 5-HT(2A) receptor (K(i)=9.0 nM) in vitro and promising in vivo characteristics.     

Synthesis of [18F]SU11248, a new potential PET tracer for imaging cancer tyrosine kinase

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-[18F]fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide, a new potential positron emission tomography tracer for imaging cancer tyrosine kinase, has been prepared by the nucleophilic substitution of the nitro-precursor N-[2-(diethylamino)ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide with K18F/Kryptofix 2.2.2 followed by a simple chromatography methodology combined solid-phase extraction with high-performance liquid chromatography purification procedures in 15-25% radiochemical yields.     

PET imaging of liposomes labeled with an [18F]-fluorocholesteryl ether probe prepared by automated radiosynthesis

A novel [¹⁸F]-labeled cholesteryl ether lipid probe was prepared by synthesis of the corresponding mesylate, which was [¹⁸F]-fluorinated by a [¹⁸F]KF, Kryptofix-222, K₂CO₃ procedure. Fluorination was done for 10 minutes at 165^°C and took place with conversion between 3 and 17%, depending on conditions. Radiolabelling of the probe and subsequent in situ purification on SEP-Paks were done on a custom-built, fully automatic synthesis robot. Long-circulating liposomes were prepared by hydration (magnetic stirring) of a lipid film containing the radiolabeled probe, followed by fully automated extrusion through 100-nm filters. The [¹⁸F]-labeled liposomes were injected into nude, tumor-bearing mice, and positron emission tomography (PET) scans were performed several times over 8 hours to investigate the in vivo biodistribution. Clear tumor accumulation, as well as hepatic and splenic uptake, was observed, corresponding to expected liposomal pharmacokinetics. The tumor accumulation 8 hours postinjection accounted for 2.25?±?0.23 (mean ± standard error of the mean) percent of injected dose per gram (%ID/g), and the tumor-to-muscle ratio reached 2.20?±?0.24 after 8 hours, which is satisfactorily high for visualization of pathological lesions. Moreover, the blood concentration was still at a high level (13.9?±?1.5 %ID/g) at the end of the 8-hour time frame. The present work demonstrates the methodology for automated preparation of radiolabeled liposomes, and shows that [¹⁸F]-labeled liposomes could be suitable as a methodology for visualization of tumors and obtaining short-term pharmacokinetics in vivo.     

Radiosynthesis and preclinical evaluation of [18F]FEM as a potential novel PET probe for tumor imaging

In the 21st century, the incidence and mortality of cancer, one of the most challenging diseases in the world, have rapidly increased. The purpose of this study was to develop 2-(2-[¹⁸F]fluoroethoxy)ethyl 4-methylbenzenesulfonate ([¹⁸F]FEM) as a positron emission tomography (PET) agent for tumor imaging. In this study, [¹⁸F]FEM was synthesized with a good radiochemical yield (45.4 ± 5.8%), high specific radioactivity (over 25 GBq/μmol), and commendable radiochemical purity (over 99%). The octanol/water partition coefficient of [¹⁸F]FEM was 1.44 ± 0.04. The probe demonstrated good stability in vitro (phosphate-buffered saline (PBS) and mouse serum (MS)), and binding specificity to five different tumor cell lines (A549, PC-3, HCC827, U87, and MDA-MB-231). PET imaging of tumor-bearing mice showed that [¹⁸F]FEM specifically accumulated at the tumor site of the five different tumor cell lines. The average tumor-to-muscle (T/M) ratio was over 2, and the maximum T/M values reached about 3.5. The biodistribution and dynamic PET imaging showed that most probes were metabolized by the liver, whereas a small part was metabolized by the kidney. Moreover, dynamic brain images and quantitative data showed [¹⁸F]FEM can quickly cross the blood brain barrier (BBB) and quickly fade out, thereby suggesting it may be a promising candidate probe for the imaging of brain tumors. The presented results demonstrated that [¹⁸F]FEM is a promising probe for tumor PET imaging.     

18F Labeled benzimidazole derivatives as potential radiotracer for positron emission tomography (PET) tumor imaging

This article reported the synthesis and bioevaluation of two [¹⁸F] labeled benzimidazole derivatives, 4-(5-(2-[¹⁸F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([¹⁸F] FNBMBBA, [¹⁸F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([¹⁸F] FEMPBBA, [¹⁸F]b1) for PET tumor imaging. The preparation [¹⁸F] FEMPBBA was completed in 1 h with overall radiochemical yield of 50–60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of both compounds were carried out, and the results are both meaningful. [¹⁸F] FEMPBBA which can be taken as a revision of [¹⁸F] FNBMBBA got an excellent result, and has significant advantages in some aspects compared with L-[¹⁸F] FET and [¹⁸F]-FDG in the same animal model, especially in tumor/brain uptake ratio. The tumor/brain uptake ratio of [¹⁸F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30 min, 60 min and 120 min, and is much higher than that of L-[¹⁸F] FET (2.54, 2.92 and 2.95) and [¹⁸F]-FDG (0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [¹⁸F] FEMPBBA is also higher than that of L-[¹⁸F] FET at 30 min and 60 min. This result indicates compound [¹⁸F] FEMPBBA is a promising radiotracer for PET tumor imaging.     

Multicellular Tumour Spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring

Background  

Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF).     

Solid-phase synthesis of 2-[18F]fluoropropionyl peptides

The 2-[(18)F]fluoropropionic (2-[(18)F]FPA) acid is used as a prosthetic group for radiolabeling proteins and peptides for targeted imaging using positron emission tomography (PET). Radiolabeling of compounds with more than one acylable functional group can lead to complex mixtures of products; however, peptides can be labeled regioselectively on the solid phase. We investigated the use of a solid-phase approach for the preparation of 2-[(18)F]fluoropropionyl peptides. [(18)F]FPA was prepared and conjugated to the peptides attached to the solid phase support. The (18)F-labeled peptides were obtained in 175 min with decay corrected yields of 10% (related to [(18)F]fluoride) and with a purity of 76-99% prior HPLC purification. The suitability of various coupling reagents and solid supports were tested for radiolabeling of several peptides of various lengths.     

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.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate: a novel radiotracer for quantifying brain butyrylcholinesterase activity by positron emission tomography

In Alzheimer's disease, cerebral cortical butyrylcholinesterase (BChE) activity is reported to be elevated. Our aim was to develop a novel (18)F-labeled tracer for quantifying cerebral BChE activity by positron emission tomography. With in vitro screening of N-[(14)C]ethylpiperidin-3- and 4-ylmethyl esters, N-[(14)C]ethylpiperidin-4-ylmethyl butyrate was selected as a lead for (18)F-labeling, affording N-[(18)F]fluoroethylpiperidin-4-ylmethyl butyrate. The (18)F-labeled butyrate showed the required properties for in vivo BChE measurement, that is, the lipophilic nature of the authentic ester, high specificity to BChE, a moderate hydrolysis rate, and the hydrophilic nature of the metabolite.     

Solid phase synthesis of [18F]labelled peptides for positron emission tomography

A strategy for the solid phase synthesis of [18F]labelled peptides has been developed. The peptides were prepared on solid support and acylated with 4-[18F]fluorobenzoic acid using HATU within 3 min and the labelled peptide was released from the solid support within 7 min. The [18F]labelled peptides were produced in good purity with a specific activity of 20-25 GBq/micromol.     

[18F]DPA-714 as a biomarker for positron emission tomography imaging of rheumatoid arthritis in an animal model

Introduction

Rheumatoid arthritis (RA) is a chronic disease, affecting 0.5 to 1% of adults in industrialized countries, in which systemic inflammation and synovitis drive joint destruction. [¹⁸F]DPA-714 is a specific tracer of the 18 kDa translocator protein (TSPO), which is overexpressed on activated macrophages, and proposed as a biomarker of neuroinflammation. Today, diagnosis of patients with early inflammatory arthritis is limited by poor sensitivity and specificity. The present study aims to investigate the potential of [¹⁸F]DPA-714 to monitor in vivo inflammatory processes at a preclinical stage via positron emission tomography (PET).

Methods

RA was induced in Dark Agouti rats by subcutaneous injection of inactivated Mycobacterium tuberculosis. Development of arthritis clinical signs was investigated daily and the severity of the disease evaluated. Animals were imaged at the peak of inflammation using [¹⁸F]DPA-714 and a small-animal PET-CT tomograph.

Results

The first clinical signs appeared at 10 days post-injection, with a peak of inflammation at 20 days. At this time, PET-analyses showed a clear uptake of [¹⁸F]DPA-714 in swollen ankles, with mean values of 0.52 ± 0.18% injected dose (ID/cc) for treated (n = 11) and 0.19 ± 0.09 for non-treated (n = 6) rats. A good correlation between [¹⁸F]DPA-714’s uptake and swelling was also found. Immunohistochemistry showed an enhanced TSPO expression in hind paws, mainly co-localized with the macrophages specific antigen CD68 expressing cells.

Conclusion

These preliminary results demonstrate that the TSPO 18kDa specific radioligand [¹⁸F]DPA-714 is adapted for the study and follow-up of inflammation linked to RA in our experimental model, suggesting also a strong potential for clinical imaging of peripheral inflammation.     

A single column,rapid quality control procedure for 6-[18F]fluoro-l-dopa and 6-[18F]fluorodopamine PET imaging agents

6-[¹⁸F]Fluoro-l-dopa and 6-[¹⁸F]fluorodopamine are promising PET imaging agents for visualizing cerebral dopaminergic centers and cardiac sympathetic innervation and function. Administration to humans requires a means to determine the purity before injection. We describe such a method using HPLC with u.v. and radioactivity detection and a single high-speed C-18 column with gradient elution. The procedure can resolve within 10 min these fluorinated catechols, their isomers, and dihydroxyphenylalanine. The chemical and radiochemical purity, and specific activity, can be determined before injection.     

Positron emission tomography imaging of cell death with [18F]FPDuramycin

The noninvasive imaging of cell death, including apoptosis and necrosis, is an important tool for the assessment of degenerative diseases and in the monitoring of tumor treatments. Duramycin is a peptide of 19-amino acids. It binds specifically to phosphatidylethanolamine a novel molecular target for cell death. N-(2-¹⁸F-Fluoropropionyl)duramycin ([¹⁸F]FPDuramycin) was prepared as a novel positron emission tomography (PET) tracer from the reaction of duramycin with 4-nitrophenyl 2-[¹⁸F]fluoropropionate ([¹⁸F]NFP). Compared with control cells (viable tumor cells), the in vitro binding of [¹⁸F]FPDuramycin with apoptotic cells induced by anti-Fas antibody resulted in a doubling increase, while the binding of [¹⁸F]FPDuramycin with necrotic cells induced by three freeze and thaw cycles resulted in a threefold increase. Biodistribution study in mice exhibited its rapid blood and renal clearance and predominant accumulation in liver and spleen over 120 min postinjection. Small-animal PET/CT imaging with [¹⁸F]FPDuramycin proved to be a successful way to visualize in vivo therapeutic-induced tumor cell death. In summary, [¹⁸F]FPDuramycin seems to be a potential PET probe candidate for noninvasive visualization of in vivo cell death sites induced by chemotherapy in tumors.