Autoradiography validation of novel tau PET tracer [F-18]-MK-6240 on human postmortem brain tissue.

[F-18]-MK-6240, a novel tau positron emission tomography (PET) tracer recently discovered for the in vivo detection of neurofibrillary tangles, has the potential to improve diagnostic accuracy in the detection of Alzheimer disease. We have examined regional and substrate-specific binding patterns as well as possible off-target binding of this tracer on human brain tissue to advance towards its validation. We applied [F-18]-MK-6240 phosphor screen and high resolution autoradiography to postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration-tau (Pick's disease, progressive supranuclear palsy and corticobasal degeneration), chronic traumatic encephalopathy, frontotemporal lobar degeneration-Tar DNA-binding protein 43 (TDP-43), dementia with Lewy bodies, cerebral amyloid angiopathy and elderly controls free of pathologic changes of neurodegenerative disease. We also directly compared the binding properties of [F-18]-MK-6240 and [F-18]-AV-1451 in human tissue, and examined potential nonspecific binding of both tau tracers to monoamine oxidases (MAO) by using autoradiography in the presence of selective monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitors. Our data indicate that MK-6240 strongly binds to neurofibrillary tangles in Alzheimer disease but does not seem to bind to a significant extent to tau aggregates in non-Alzheimer tauopathies, suggesting that it may have a limited utility for the in vivo detection of these pathologies. There is no evidence of binding to lesions containing ß-amyloid, α-synuclein or TDP-43. In addition, we identified MK-6240 strong off-target binding to neuromelanin and melanin-containing cells, and some weaker binding to areas of hemorrhage. These binding patterns are nearly identical to those previously reported by our group and others for [F-18]-AV-1451. Of note, [F-18]-MK-6240 and [F-18]-AV-1451 autoradiographic binding signals were only weakly displaced by competing concentrations of selective MAO-B inhibitor deprenyl but not by MAO-A inhibitor clorgyline, suggesting that MAO enzymes do not appear to be a significant binding target of any of these two tracers. Together these novel findings provide relevant insights for the correct interpretation of in vivo [F-18]-MK-6240 PET imaging.

Radiosynthesis and modified quality control of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) for brain tumor imaging.

O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) is the most promising radio-labeled amino acid tracer for brain tumor imaging due to the limitation of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and L-methyl-[11C]methionine (11C-MET). However, it has some limitations in radiosynthesis and related quality control that make it less frequently used in many PET centers, in this study, we report a new modification of [18F]FET production using a commercially available fully automated GRP SCINTOMICS module overcoming some of the existing limitations along with a suggestion of a simplified quality control procedure with special focus placed on enantiomeric and radiochemical purity. ([18F]FET) was produced in high radiochemical and enantiomeric purity more than 99% and non-decay corrected yield 25±5% in about 55min.

Synthesis and preliminary in vivo evaluation of new [18F]fluoro-inositols as Positron Emission Tomography radiotracers.

This study describes the synthesis and radiosynthesis of eight new [18F]fluoro-inositol-based radiotracers in myo- and scyllo-inositol configuration. These radiotracers are equipped with a propyl linker bearing fluorine-18. This fluorinated arm is either on a hydroxyl group, i.e. O-alkylated inositols, or on the cyclohexyl backbone, i.e. C-branched derivatives. To modulate lipophilicity, inositols were synthesized in acetylated or hydroxylated form. Automated radiosynthesis was performed on the AllInOne module and the radiotracers were produced in good radiochemical yields (15-31.5% dc). Preliminary in vivo preclinical evaluation of these eight [18F]fluoro-inositols as Positron Emission Tomography (PET) imaging agents in a breast tumour-bearing mouse model was performed and compared with [18F]-2-fluoro-2-deoxy-d-glucose ([18F]FDG). Amongst the different inositols, [18F]myo-2 showed the highest tumour uptake 2.34±0.39%ID/g, revealing the potential of this tracer for monitoring breast cancer.

Fully automated one-pot two-step synthesis of 4-[(18)F]-ADAM, a potent serotonin transporter imaging agent.

INTRODUCTION: N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine (4-[(18)F]-ADAM, 2) is a potent serotonin transporter (SERT) imaging agent. In order to fulfill the demand of clinical studies, we have developed a fully automated one-pot two-step synthesis of this potent SERT imaging agent. METHODS: The 4-[(18)F]-ADAM (2) was synthesized using a commercially available GE TRACERlab FN module. Briefly, the precursor, N,N-dimethyl-2-(2,4-dinitrophenylthio) benzylamine (1) in DMSO was reacted with K[(18)F]/K2.2.2 in a glassy carbon reaction vessel at 120°C for 7.5min followed by reduction of the intermediate with NaBH4/Cu(OAc)2 in EtOH in the same vessel at 80°C for 20min. The reaction mixture was then purified with high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) to give (2). The quality of (2) synthesized by this method was verified by HPLC and TLC as compared to its authentic sample synthesized by two-pot two-step method. RESULTS: Using this automated one-pot two-step method, the radiochemical yield (RCY) of (2) was 2.5±0.8% (n=12, EOS) in a synthesis time of 100±6min from EOB with a specific activity of 4.4±1.9Ci/µmol (n=12, EOS). Radioligand (2) was stable over 4h at room temperature. CONCLUSIONS: This fully automated one-pot two-step synthetic method using a commercially available GE TRACERlab FN module could simplify the synthesis of 4-[(18)F]-ADAM (2) and fulfill its demand for both animal and human studies, especially for study sites without a cyclotron.

Radionuclidic purity tests in (18)F radiopharmaceutIcals production process.

Radionuclidic purity tests of (18)F radiopharmaceuticals (Na(18)F and fluorodeoxyglucose [(18)F]FDG) and radionuclide composition analysis of irradiated water [(18)O]H2O were performed. The measurements were conducted using a High-Purity Germanium (HPGe) detector and a liquid scintillation counter. Radionuclide identification and activity measurements were performed for samples from different stages of the production process. Most of the impurities were detected on QMA (quaternary methylammonium) anion exchange columns and in liquid wastes. Using liquid scintillation counting, the activity of (3)H resulting from the (18)O[p, (3)H](16)O reaction was determined. It was shown that all of the impurities were efficiently determined and eliminated in the radiopharmaceuticals synthesis process and that the final products meet the requirements set by relevant regulations.

Automated production of [¹8F]VAT suitable for clinical PET study of vesicular acetylcholine transporter.

Automated production of a promising radiopharmaceutical (-)-(1-(8-(2-[(18)F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)(4-fluorophenyl)methanone ([(18)F]VAT) for the vesicular acetylcholine transporter(VAChT) was achieved using a two-step procedure in a current Good Manufacturing Practices fashion. The production of [(18)F]VAT was accomplished in approximately 140 min, with radiochemical yield of ~15.0% (decay corrected), specific activity>111 GBq/µmol, radiochemical purity>99% and mass of VAT ~3.4 µg/batch (n>10). The radiopharmaceutical product meets all quality control criteria for human use, and is suitable for clinical PET studies of VAChT.

Traceability from governmental producers of radiopharmaceuticals in measuring (18)F in Brazil.

Since the inception of its proficiency test program to evaluate radionuclide measurement in hospitals and clinics, the National Metrology Laboratory of Ionizing Radiation-LNMRI, that represents Brazilian National Metrology Institute (NMI) for ionizing radiation has expanded its measurement and calibration capability. Requirements from the National Health Surveillance Agency from Ministry of Health (ANVISA), to producers of radiopharmaceuticals provided an opportunity to improve the full traceability chain to the highest level. Fluorodeoxyglucose (FDG-(18)F) is the only radiopharmaceutical simultaneously produced by all Brazilian radiopharmaceutical production centers (RPCs). By running this proficiency test, LNMRI began to provide them with the required traceability. For evaluation, the ratio of RPC to reference value results and ISO/IEC17043:2010 criteria were used. The reference value established as calibration factor on the secondary standard ionization chamber was obtained from three absolute measurements systems, and routinely confirmed in each round of proficiency test by CIEMAT/NIST liquid scintillation counting. The γ-emitting impurities were checked using a High-Purity Germanium (HPGe) detector. The results show that Brazilian RPCs are in accordance with (accuracy within ±10%) the Brazilian standard for evaluation of measurements with radionuclide calibrators (CNEN NN 3.05., 2013). Nevertheless, the RPCs should improve the methodology of uncertainty estimates, essential when using the statistical criteria of ISO/IEC 17043 standard, in addition to improving accuracy to levels consistent with their position in the national traceability chain.

Comparison of (18)F activity measurements at the VNIIM, NPL and the ENEA-INMRI using the SIRTI of the BIPM.

In 2014, the first three comparisons of activity measurements of (18)F were carried out at the VNIIM, NPL and the ENEA-INMRI using the BIPM's Transfer Instrument of the International Reference System. The transfer instrument and the NMIs primary measurement methods are briefly described. The degrees of equivalence with the key comparison reference value defined in the frame of the corresponding SIR comparison have been evaluated. World-wide consistency of activity measurements of (18)F is demonstrated.

Impact of Recent Change in the National Institute of Standards and Technology Standard for 18F on the Relative Response of 68Ge-Based Mock Syringe Dose Calibrator Standards.

UNLABELLED: As a result of a recent change in the National Institute of Standards and Technology (NIST) activity standard for (18)F, we have determined new relative response ratios for a (68)Ge solid epoxy mock syringe source used in activity calibrators as a long-lived substitute for (18)F. New standardized solutions of each radionuclide were used to determine the response ratios while maintaining traceability to national standards. This work updates our previously published data from 2010. METHODS: Following our previously published methodology, solution-filled mock syringe sources, identical in geometry to the solid (68)Ge epoxy calibration source currently on the market, were prepared using NIST-calibrated solutions of (68)GeCl4 and (18)F-FDG and directly compared in several models of activity calibrators to determine empirically the relative response ratios for these 2 radionuclides. RESULTS: The new relative response ratios measured in this study reflect the change in (18)F activity measurements that arise from the recent -4% change in the NIST activity standard. The results allow the (68)Ge activity of the mock syringe source to be expressed in terms of equivalent (18)F activity, with a relative combined standard uncertainty of about 0.8% for the activity calibrators used in this study. CONCLUSION: This work revises our previously derived relative response ratios for (18)F and (68)Ge by -3.7%, allowing users of the commercial mock syringe surrogate source to calibrate their activity calibrators in a way that is consistent with the recent change in the NIST (18)F standard.

Use of LC-MS for the quality control of radiopharmaceuticals: example of [(18)F]ML10.

[(18)F]ML10 is a promising novel low molecular weight positron emission tomography probe for apoptosis. As part of the quality control to support clinical studies for cancer therapy monitoring in the GSK Clinical Imaging Centre, a simple and sensitive liquid chromatography mass spectrometry method has been developed and validated for the quantification of total ML10 and impurity content in the final product. Chromatographic separation of ML10 and its radiolabelling precursor and impurities was achieved. Mass curves were constructed from a concentration range of ML10 and known impurities and were linear. Quantification was achieved by comparison of the area under the curve for ML10 content (m/z = 205) and the mass curve. The method was validated over a concentration range of 0.1-1 µg/ml.

Current status and future needs for standards of radionuclides used in positron emission tomography.

Positron Emission Tomography (PET) is being increasingly used as a quantitative technique for detecting disease and monitoring patient progress during treatment. To ensure the validity of the quantitative information derived from the imaging data, it is imperative that all radioactivity measurements that are part of the imaging procedure be traceable to national or international standards. This paper reviews the current status of standards for positron emitting radionuclides (e.g., (18)F, (68)Ge/(68)Ga, and (124)I) and suggests needs for future work.

Standardization of 18F by digital coincidence counting.

The radioactivity of (18)F has been measured by a digital coincidence counting (DCC) system. The main advantages of the digital coincidence counting technique are a shortening of the measurement time as compared with conventional coincidence counting and an ability to obtain activities with various experimental parameters through off-line analysis. The measurement results of radioactivity for (18)F solution were compared with those of a conventional coincidence counting technique and a reference ion chamber method.

Radioactivity measurement of 18F in 16 ml vials for calibration of radionuclide calibrators.

Fluorine-18 is obtained through the reaction (18)O(p, n)(18)F using a cyclotron that is situated in a hospital in Jakarta. Standardization of the (18)F solution is performed by gamma spectrometry using calibration sources of (152)Eu, (60)Co and (137)Cs that have traceability to the International System of units (SI). The activities in the 16 ml vials that were used for calibrating the radionuclide calibrators were between 1 and 2 GBq, with expanded uncertainties of 3.8%. The expanded uncertainty, at a coverage factor of k=2, on the derived calibration factor for the radionuclide calibrator was 6.6%.

FLT-PET may not be a reliable indicator of therapeutic response in p53-null malignancy.

FDG (18F-deoxy-glucose) is the current gold standard for PET imaging. FLT (3'-deoxy-3'-(18F-fluorothymidine), a PET imaging marker of proliferation, has been proposed as an alternative to FDG for the assessment of therapeutic response. We examined the therapeutic predictive value of FLT-PET and FDG-PET using CALU-6, a human, p53-null, non-small cell lung cancer cell line with comparison of combined targeted therapy, TRAIL and sorafenib, versus combined conventional chemotherapy, docetaxel and cisplatin. CALU-6 tumor-bearing nu/nu mice (n=46) were evaluated in 3 therapeutic trials measuring FLT and FDG prediction of tumor response at 72 h following initiation of daily combination therapy with targeted agents, TRAIL (200 µg i.v.) and sorafenib (30 mg/kg i.p.) and compared to conventional chemotherapeutics cisplatin (3 mg/kg i.p.) and docetaxel (7.5 mg/kg i.p.). PET imaging response was compared to morphological and histological indicators of therapeutic response, including decreased vascularity (in vivo AngioSense imaging and anti-CD31 staining), slowed tumor growth (caliper measurements), decreased cellular proliferation (Ki-67 staining) and increased apoptosis (TUNEL staining). Decreases in tumor accumulation of FLT (FLTMAX -30%, p=0.03) at 72 h post treatment were observed in response to TRAIL and sorafenib combination therapy resulting in smaller, less vascular, more apoptotic tumors. No similar reduction in tumor accumulation of FLT (FLTMAX -2%, p=0.67) was observed 72 h following initiation of cisplatin and docetaxel combination therapy, despite histological and morphological evidence of drug response. In contrast, tumor imaging with FDG did demonstrate a decrease in accumulation in both treatment groups, -21% (p=0.30) in response to cisplatin/docetaxel and -8% (p=0.59) in response to TRAIL/sorafenib, but did not reach statistical significance. FLT, but not FDG, is predictive of therapeutic response to the targeted regimen TRAIL/sorafenib. However, FLT-PET may not predict therapeutic response to DNA damaging agents in p53-null tumors, likely due to loss of cell cycle control of thymidine kinase 1 (TK1). Thus, tumor imaging response by FLT may be limited in human tumors without functional p53.

Standardization of fluorine-18 manufacturing processes: new scientific challenges for PET.

In [(18)F]fluoride chemistry, the minute amounts of radioactivity taking part in a radiolabeling reaction are easily outnumbered by other reactants. Surface areas become comparably larger and more influential than in standard fluorine chemistry, while leachables, extractables, and other components that normally are considered small impurities can have a considerable influence on the efficiency of the reaction. A number of techniques exist to give sufficient (18)F-tracer for a study in a pre-clinical or clinical system, but the chemical and pharmaceutical understanding has significant gaps when it comes to scaling up or making the reaction more efficient. Automation and standardization of [(18)F]fluoride PET tracers is a prerequisite for reproducible manufacturing across multiple PET centers. So far, large-scale, multi-site manufacture has been established only for [(18)F]FDG, but several new tracers are emerging. In general terms, this transition from small- to large-scale production has disclosed several scientific challenges that need to be addressed. There are still areas of limited knowledge in the fundamental [(18)F]fluoride chemistry. The role of pharmaceutical factors that could influence the (18)F-radiosynthesis and the gaps in precise chemistry knowledge are discussed in this review based on a normal synthesis pattern.

Quantifying and reducing the effect of calibration error on variability of PET/CT standardized uptake value measurements.

UNLABELLED: The purpose of this study was to measure the errors introduced by regular calibration of PET/CT scanners and to minimize the effect of calibration error on standardized uptake value measurements. METHODS: Global calibration factors from 2 PET/CT scanners were recorded for 3.5 and 1.8 y, comparing manufacturer-recommended protocols with modified protocols to evaluate error contributions due to operator-influenced procedures. Dose calibrator measurements were evaluated using National Institute of Standards and Technology-traceable sources. RESULTS: Dose calibrator variability was less than 1%, although there was a consistent bias. Global scaling variability was reduced from 6% to 4% for scanner 1 and from 11% to 4% for scanner 2 when quality assurance and quality control procedures were applied to the calibration protocol. When calibrations were done using a (68)Ge/(68)Ga phantom, the variability for both scanners was reduced to approximately 3%. CONCLUSION: Applying quality assurance and quality control procedures to scanner calibration reduces variability, but there is a still a residual longitudinal scanner variability of 3%-4%. The procedures proposed here reduce the impact of operator error on scanner calibration and thereby minimize longitudinal variability in standardized uptake value measurements.

An efficient and aseptic preparation of "sodium fluoride ((18)F) injection" in a GMP compliant facility.

INTRODUCTION: "Sodium fluoride ((18)F) injection" is an isotonic NaCl solution containing [(18)F]NaF to be used as bone imaging agent. Although its NDA was approved by the US FDA in 1972, it has not been commercially available since 1975 due to mostly the popularity of (99m)Tc-MDP. Recently, advances in PET/CT technology and the often interrupted (99m)Tc supply have led to the renewed interest in the use of [(18)F]NaF to detect bone metastases in cancer patients. This report introduces an efficient, low-cost and aseptic preparation of "Sodium fluoride ((18)F) injection" for PET scan. METHOD: (18)F-Fluoride in target water from cyclotron was adsorbed onto four different forms of anion-exchange resins then desorbed by isotonic NaCl solution into the product vial. One of the resins that yielded the product at the suitable pH was used for the aseptic preparation. The components for this setup, including stopcocks, extension tubes, etc., were all single-use, individually packed and sterile. The process was done in a lead-line isolator maintained in grade A (PIC/S) aseptic condition. The quality of the obtained "Sodium fluoride ((18)F) injection" was analyzed according to its monograph in the European Pharmacopoeia (EP). RESULTS: The resin in the chloride form yielded the product of pH 6.7 and was chosen for the subsequent preparation. The radiochemical yield was quantitative. The product met all criteria specified in EP, including biological, physical and chemical specifications. CONCLUSIONS: This method is an efficient, space-saving and extremely low-cost operation that easily performed in an aseptic environment meeting GMP standard. The quality of the "Sodium fluoride ((18)F) injection" so yielded meets EP specifications. This setup provides hospital with facility meeting GMP standard a cost effective and efficient method for "Sodium fluoride ((18)F) injection" production without the need for the expensive automatic module and extra QC instrument.

Standardisation of 18F by a coincidence method using full solid angle detectors.

A solution of (18)F was standardised with a 4pibeta-4pigamma coincidence counting system in which the beta detector is a one-inch diameter cylindrical UPS89 plastic scintillator, positioned at the bottom of a well-type 5''x5'' NaI(Tl) gamma-ray detector. Almost full detection efficiency-which was varied downwards electronically-was achieved in the beta-channel. Aliquots of this (18)F solution were also measured using 4pigamma NaI(Tl) integral counting and Monte Carlo calculated efficiencies as well as the CIEMAT-NIST method. Secondary measurements of the same solution were also performed with an IG11 ionisation chamber whose equivalent activity is traceable to the Système International de Référence through the contribution IRA-METAS made to it in 2001; IRA's degree of equivalence was found to be close to the key comparison reference value (KCRV). The (18)F activity predicted by this coincidence system agrees closely with the ionisation chamber measurement and is compatible within one standard deviation of the other primary measurements. This work demonstrates that our new coincidence system can standardise short-lived radionuclides used in nuclear medicine.

Activity standardisation of (18)F and ionisation chamber calibration for nuclear medicine.

Primary activity standardisations were performed on solutions of (18)F using 4pibeta-gamma coincidence counting and liquid scintillation counting (LSC) according to the CIEMAT/NIST method. A beta(+)-emission probability of 96.86% was used for both methods. The various standardised (18)F solutions were measured in ionisation chambers of the Physikalisch-Technische Bundesanstalt (PTB) and compared by determining radionuclide calibration factors. Already in 2001 an (18)F solution had been standardised at the PTB and compared with the results of nine national metrology institutes (NMIs), using the ISOCAL IV secondary radionuclide calibrators of the National Physical Laboratory (NPL) as transfer instruments and a (68)Ge check source solution. These results were linked to the International Reference System (SIR) at the Bureau International des Poids et Mesures (BIPM) by aliquots of solutions sent by the Laboratoire National Henri Becquerel (BNM-LNHB) and the NPL. Further on, in 2005, PTB sent an aliquot of an (18)F solution to the SIR for ionisation chamber measurements. A value of the equivalent activity was determined and included in the key comparison database (KCDB). The recent PTB value of the equivalent activity of the SIR is in good agreement with the key comparison reference value determined from five NMIs. These results confirm that the standardisation of (18)F solutions can be achieved with the accuracy required for use in nuclear medicine and, in particular, for applications in positron emission tomography (PET).

Standardization of 18F by coincidence and LSC methods.

The nuclide 18F disintegrates to 18O by beta+ emission (96.86%) and electron capture (3.14%) with a half-life of 1.8288 h. It is widely used in nuclear medicine for positron emission tomography (PET). A radioactive solution of this nuclide has been standardized by two techniques: coincidence measurements with a pressurized proportional counter and liquid scintillation counting using the CIEMAT/NIST method. One ampoule containing a solution calibrated in activity was sent for measurement at the International Reference System maintained by the BIPM. Results are in excellent agreement with SIR values.