The First Human Application of an F-18-Labeled Tryptophan Analog for PET Imaging of Cancer.

PURPOSE: Preclinical studies showed the tryptophan analog PET radiotracer 1-(2-18F-fluoroethyl)-L-tryptophan (18F-FETrp) to accumulate in various tumors, including gliomas, and being metabolized via the immunosuppressive kynurenine pathway. In this first-in-human study, we tested the use 18F-FETrp-PET in patients with neuroendocrine and brain tumors. PROCEDURES: We applied dynamic brain imaging in patients with gliomas (n = 2) and multi-pass 3D whole-body PET scans in patients with neuroendocrine tumors (n =4). Semiquantitative analysis of organ and tumor tracer uptake was performed using standardized uptake values (SUVs). In addition, organ dosimetry was performed based on extracted time-activity curves and the OLINDA software. RESULTS: Neuroendocrine tumors showed an early peak (10-min post-injection) followed by washout. Both gliomas showed prolonged 18F-FETrp accumulation plateauing around 40 min and showing heterogeneous uptake including non-enhancing tumor regions. Biodistribution showed moderate liver uptake and fast clearance of radioactivity into the urinary bladder; the estimated effective doses were similar to other 18F-labeled radioligands. CONCLUSIONS: The study provides proof-of-principle data for the safety and potential clinical value of 18F-FETrp-PET for molecular imaging of human gliomas.

Automated radiosynthesis of 1-(2-[18 F]fluoroethyl)-L-tryptophan ([18 F]FETrp) for positron emission tomography (PET) imaging of cancer in humans.

The radiotracer 1-(2-[18 F]fluoroethyl)-L-tryptophan (L-[18 F]FETrp or [18 F]FETrp) is a substrate of indoleamine 2,3-dioxygenase, the initial and key enzyme of the kynurenine pathway associated with tumoral immune resistance. In preclinical positron emission tomography studies, [18 F]FETrp is highly accumulated in a wide range of primary and metastatic cancers, such as lung cancer, prostate cancer, and gliomas. However, the clinical translation of this radiotracer into the first-in-human trial has not been reported, partially due to its racemization during radiofluorination which renders the purification of the final product challenging. However, efficient purification is essential for human studies in order to assure radiochemical and enantiomeric purity. In this work, we report a fully automated radiosynthesis of [18 F]FETrp on a Synthra RNPlus research module, including a one-pot two steps radiosynthesis, dual independent chiral and reverse-phase semipreparative high-performance liquid chromatography purifications, and solid-phase extraction-assisted formulation. The presented approach has led to its Investigational New Drug application and approval that allows the testing of this tracer in humans.

HPLC-free and cassette-based nucleophilic production of [18F]FDOPA for clinical use.

Radiotracer 3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine (L-6-[18F]fluorodopa or [18F]FDOPA) is widely used for PET imaging of dopamine metabolism in several diseases including Parkinson's Disease, brain tumor, neuroendocrine tumors, and focal hyperinsulinism of infancy. In 2019, [18F]FDOPA was approved by US FDA for detection of dopaminergic nerve terminals in the striatum of adult patients with suspected Parkinsonian Syndromes. A convenient and reliable method is desired for fully automated production of [18F]FDOPA under cGMP compliance to meet the increasing clinical need. In this study, we reported a cassette-based automated production of [18F]FDOPA using a GE Fastlab 2 module and the quality control (QC) under fully cGMP compliant environment. Briefly, automated radiosynthesis of [18F]FDOPA was processed via nucleophilic radio-fluorination using FDOPA Fastlab cassette and solid phase extraction (SPE) purification. The QC tests of [18F]FDOPA, including appearance, pH, half-life, radiochemical purity and identity, enantiomeric purity, chemical impurities, molecular activity, radioactive concentration, filter integrity, endotoxin, and sterility, were conducted at the end of synthesis (EOS) and 8 h after EOS during the validation runs. Three consecutive productions of [18F]FDOPA were reliably achieved with desired radiochemical yield and high radiochemical/enantiomeric purities and molar activity. The uncorrected radiochemical yields of [18F]FDOPA were 9.3-9.8% with a total synthesis time of ~140 min. Both radiochemical and enantiomeric purities of [18F]FDOPA were >99.9% and the molar activities were 2.1-3.9 Ci/µmole at EOS. The full QC results at EOS and 8 h after EOS showed that the produced [18F]FDOPA met all release criteria for clinical use within 8 hours of expiration time. Three consecutive validation runs and QC results demonstrated the efficacy of cassette-based production of [18F]FDOPA for routine clinical use.

Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging.

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection.

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and 125I radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (99mTc)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and 125I), which overlapped well with areas of tumor cell death (99mTc-duramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and 99mTc-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and 99mTc-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

New transgenic NIS reporter rats for longitudinal tracking of fibrogenesis by high-resolution imaging.

Fibrogenesis is the underlying mechanism of wound healing and repair. Animal models that enable longitudinal monitoring of fibrogenesis are needed to improve traditional tissue analysis post-mortem. Here, we generated transgenic reporter rats expressing the sodium iodide symporter (NIS) driven by the rat collagen type-1 alpha-1 (Col1α1) promoter and demonstrated that fibrogenesis can be visualized over time using SPECT or PET imaging following activation of NIS expression by rotator cuff (RC) injury. Radiotracer uptake was first detected in and around the injury site day 3 following surgery, increasing through day 7-14, and declining by day 21, revealing for the first time, the kinetics of Col1α1 promoter activity in situ. Differences in the intensity and duration of NIS expression/collagen promoter activation between individual RC injured Col1α1-hNIS rats were evident. Dexamethasone treatment delayed time to peak NIS signals, showing that modulation of fibrogenesis by a steroid can be imaged with exquisite sensitivity and resolution in living animals. NIS reporter rats would facilitate studies in physiological wound repair and pathological processes such as fibrosis and the development of anti-fibrotic drugs.

[18F]Tetrafluoroborate ([18F]TFB) and its analogs for PET imaging of the sodium/iodide symporter.

Sodium/iodide symporter (NIS)-mediated iodide uptake in thyroid follicular cells is the basis of clinical utilization of radioiodines. The cloning of the NIS gene enabled applications of NIS as a reporter gene in both preclinical and translational research. Non-invasive NIS imaging with radioactive iodides and iodide analogs has gained much interest in recent years for evaluation of thyroid cancer and NIS reporter expression. Although radioiodines and [99mTc]pertechnetate ([99mTc]TcO4-) have been utilized in positron emission tomography (PET) and single photon emission computed tomography (SPECT), they may suffer from limitations of availability, undesirable decay properties or imaging sensitivity (SPECT versus PET). Recently, [18F]tetrafluoroborate ([18F]TFB or [18F]BF4-) and other fluorine-18 labeled iodide analogs have emerged as a promising iodide analog for PET imaging. These fluorine-18 labeled probes have practical radiosyntheses and biochemical properties that allow them to closely mimic iodide transport by NIS in thyroid, as well as in other NIS-expressing tissues. Unlike radioiodides, they do not undergo organification in thyroid cells, which results in an advantage of relatively lower uptake in normal thyroid tissue. Initial clinical trials of [18F]TFB have been completed in healthy human subjects and thyroid cancer patients. The excellent imaging properties of [18F]TFB for evaluation of NIS-expressing tissues indicate its bright future in PET NIS imaging. This review focuses on the recent evolution of [18F]TFB and other iodide analogs and their potential value in research and clinical practice.

Synthesis and evaluation of 18F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model.

Noninvasive imaging of iodide uptake via the sodium/iodide symporter (NIS) has received great interest for evaluation of thyroid cancer and reporter imaging of NIS-expressing viral therapies. In this study, we investigate 18F-labeled hexafluorophosphate (HFP or PF6-) as a high-affinity iodide analog for NIS imaging. 18F-HFP was synthesized by radiofluorination of phosphorus pentafluoride·N-methylpyrrolidine complex and evaluated in human NIS (hNIS)-expressing C6 glioma cells and a C6 glioma xenograft mouse model. 18F-HFP was obtained in radiochemical yield of 10 ±â€¯5%, radiochemical purity of >96% and specific radioactivity of 604 ±â€¯18 MBq/µmol. Specific uptake of 18F-HFP and high affinity of 19F-HFP were observed in hNIS+ C6-glioma cells. PET imaging showed robust uptake of 18F-HFP in NIS-expressing tissues (thyroid, stomach, and hNIS+ C6 glioma xenografts), and the uptake of 18F-HFP was blocked by NaClO4 pretreatment. Specific accumulation in hNIS-expressing xenograft (hNIS+) was observed relative to isogenic control tumor (hNIS-). Clearance of 18F-HFP was predominantly through renal excretion. The biodistribution showed consistent results with PET imaging. Minimal bone uptake was observed over 2 h period post-injection, indicating excellent in vivo stability of 18F-HFP. Although improvement in specific radioactivity is desirable, the results indicate that 18F-HFP is a promising candidate radiotracer for further evaluation for NIS imaging.

Safety, pharmacokinetics, metabolism and radiation dosimetry of 18F-tetrafluoroborate (18F-TFB) in healthy human subjects.

BACKGROUND: 18F-Tetrafluoroborate (18F-TFB) is a promising iodide analog for PET imaging of thyroid cancer and sodium/iodide symporter (NIS) reporter activity in viral therapy applications. The aim of this study was to evaluate the safety, pharmacokinetics, biodistribution, and radiation dosimetry of high-specific activity 18F-TFB in healthy human subjects. METHODS: 18F-TFB was synthesized with specific activity of 3.2 ± 1.3 GBq/µmol (at the end of synthesis). Dynamic and whole-body static PET/CT scans over 4 h were performed after intravenous administration of 18F-TFB (333-407 MBq) in four female and four male healthy volunteers (35 ± 11 years old). Samples of venous blood and urine were collected over the imaging period and analyzed by ion-chromatography HPLC to determine tracer stability. Vital signs and clinical laboratory safety assays were measured to evaluate safety. RESULTS: 18F-TFB administration was well tolerated with no significant findings on vital signs and no clinically meaningful changes in clinical laboratory assays. Left-ventricular blood pool time-activity curves showed a multi-phasic blood clearance of 18F-radioactivity with the two rapid clearance phases over the first 20 min, followed by a slower clearance phase. HPLC analysis showed insignificant 18F-labeled metabolites in the blood and urine over the length of the study (4 h). High uptakes were seen in the thyroid, stomach, salivary glands, and bladder. Urinary clearance of 18F-TFB was prominent. Metabolic stability was evidenced by low accumulation of 18F-radioactivity in the bone. Effective doses were 0.036 mSv/MBq in males and 0.064 mSv/MBq in females (p = 0.08, not significant). CONCLUSIONS: This initial study in healthy human subjects showed 18F-TFB was safe and distributed in the human body similar to other iodide analogs. These data support further translational studies with 18F-TFB as NIS gene reporter and imaging biomarker for thyroid cancer and other disease processes that import iodide.

Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type 1.

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase-deficient (Fah(-/-)) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah(-/-) pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH(+) cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah(-/-) liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah(-/-) pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.

Synthesis of 18F-Tetrafluoroborate via Radiofluorination of Boron Trifluoride and Evaluation in a Murine C6-Glioma Tumor Model.

UNLABELLED: The sodium/iodide symporter (NIS) is under investigation as a reporter for noninvasive imaging of gene expression. Although (18)F-tetrafluoroborate ((18)F-TFB, (18)F-BF4 (-)) has shown promise as a PET imaging probe for NIS, the current synthesis method using isotopic exchange gives suboptimal radiochemical yield and specific activity. The aim of this study was to synthesize (18)F-TFB via direct radiofluorination on boron trifluoride (BF3) to enhance both labeling yield and specific activity and evaluation of specific activity influence on tumor uptake. METHODS: An automated synthesis of (18)F-TFB was developed whereby cyclotron-produced (18)F-fluoride was trapped on a quaternary methyl ammonium anion exchange cartridge, then allowed to react with BF3 freshly preformulated in petroleum ether/tetrahydrofuran (50:1). The resultant (18)F-TFB product was retained on the quaternary methyl ammonium anion exchange cartridge. After the cartridge was rinsed with tetrahydrofuran and water, (18)F-TFB was eluted from the cartridge with isotonic saline, passing through 3 neutral alumina cartridges and a sterilizing filter. Preclinical imaging studies with (18)F-TFB were performed in athymic mice bearing NIS-expressing C6-glioma subcutaneous xenografted tumors to determine the influence of specific activity on tumor uptake. RESULTS: Under optimized conditions, (18)F-TFB was synthesized in a radiochemical yield of 20.0% ± 0.7% (n = 3, uncorrected for decay) and greater than 98% radiochemical purity in a synthesis time of 10 min. Specific activities of 8.84 ± 0.56 GBq/µmol (n = 3) were achieved from starting (18)F-fluoride radioactivities of 40-44 GBq. An avid uptake of (18)F-TFB was observed in human NIS (hNIS)-expressing C6-glioma xenografts as well as expected NIS-mediated uptake in the thyroid and stomach. There was a positive correlation between the uptake of (18)F-TFB in hNIS-expressing tumor and specific activity. CONCLUSION: A rapid, practical, and high-specific-activity synthesis of the NIS reporter probe (18)F-TFB was achieved via direct radiofluorination on BF3 using an automated synthesis system. The synthesis of high-specific-activity (18)F-TFB should enable future clinical studies with hNIS gene reporter viral constructs.

Production and Transport of Gaseous 18F-Synthons: 18F-Acyl Fluorides.

Fluorine-18 (18F, T 1/2=109.7 min) is a positron-emitting isotope that has found extensive application as a radiolabel for positron emission tomography (PET). Although gaseous 11C-CO2 and 11C-CH4 are practically transported from cyclotron to radiochemistry processes, 18F-fluoride is routinely transported in aqueous solution because it is commonly produced by proton irradiation of 18O-enriched water. In most cases, subsequent dry-down steps are necessary to prepare reactive 18F-fluoride for radiofluorination. In this work, a simple module was designed to generate gaseous 18F-acyl fluorides from aqueous 18F-fluoride solution by solid phase 18F-radiofluorination of acyl anhydride. The gaseous 18F-acyl fluorides were purified through a column containing Porapak Q/Na2SO4, resulting in high yields (>86%), purities (>99%) and specific activities (>1200 GBq/µmol). Prototypic 18F-acetyl fluoride (18F-AcF) was readily transported through 15 m of 0.8 mm ID polypropylene tubing with low (0.64 ± 0.12 %) adsorption to the tubing. Following dissolution of 18F-AcF in solvent containing base, highly reactive 18F-flouride was generated immediately and used directly for 18F-labeling reactions. These data indicate that 18F-acyl fluorides represent a new paradigm for preparation and transport of anhydrous, reactive 18F-fluoride for radiofluorinations.