Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module

We developed a new fully automated method for the synthesis of [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2′-nitro-1′-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [¹⁸F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105°C for 360 s, followed by heating at 75°C for 280 s and hydrolysis with 1 N HCl at 105°C for 300 s. Using 3.7 GBq of [¹⁸F]F⁻ as a starting activity, [¹⁸F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5±3.5% for 60.0±5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5±2.8% (337±25 GBq/μmol) for 70.0±3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4±2.9% and 52.8±4.2%, respectively (n=3). The solid-phase purification removed unreacted [¹⁸F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2±1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.     

A robotic synthesis of [18F]fluoromisonidazole ([18F]FMISO).

This study aimed to develop an automated synthesis of [18F]fluoromisonidazole ([18F]FMISO) using a Scanditronix Anatech RB III robotic system. [18F]HF was produced via the 18O(p,n)18F reaction using a Scanditronix MC17F cyclotron. On average, a typical run produced [18F]FMISO with an uncorrected radiochemical yield of 30+/-5% at end of synthesis (EOS) from the irradiation of 95% enriched [18O]water. The total synthesis time was 65 min. The retention time of [18F]FMISO (the radio-peak) was 4.9 min, which was consistent with the authentic FMISO (the ultraviolet peak). The radiochemical purity was greater than 97%. Preparation of [18F]FMISO using the automated robotic system is highly reliable and reproducible, and the radiation burden for the operator can be largely reduced. Sufficient radioactivities of [18F]FMISO could be obtained for non-invasive tumor hypoxia imaging in vivo with positron emission tomography (PET).     

Fully automated synthesis system of 3'-deoxy-3'-[18F]fluorothymidine

We developed a new fully automated method for the synthesis of 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), by modifying a commercial FDG synthesizer and its disposable fluid pathway. Optimal labeling condition was that 40 mg of precursor in acetonitrile (2 mL) was heated at 150 degrees C for 100 sec, followed by heating at 85 degrees C for 450 sec and hydrolysis with 1 N HCl at 105 degrees C for 300 sec. Using 3.7 GBq of [18F]F- as starting activity, [18F]FLT was obtained with a yield of 50.5 +/- 5.2% (n = 28, decay corrected) within 60.0 +/- 5.4 min including HPLC purification. With 37.0 GBq, we obtained 48.7 +/- 5.6% (n = 10). The [18F]FLT showed the good stability for 6 h. This new automated synthesis procedure combines high and reproducible yields with the benefits of a disposable cassette system.     

Fully automated [18F]fluorocholine synthesis in the TracerLab MX FDG Coincidence synthesizer

INTRODUCTION: We developed a new fully automated method for the radiosynthesis of [18F]fluorocholine by modifying the commercial 2-[18F]fluoro-2-d-deoxy-glucose ([18F]FDG) synthesizer module (GE TracerLab MX, formerly Coincidence). METHODS: [18F]Flurocholine was synthesized by (18)F-fluoroalkylation of N,N-dimethylaminoethanol using [18F]fluorobromomethane as fluoromethylating agent. [18F]Fluorobromomethane was produced by reaction of dibromomethane with [18F]fluoride, assisted by Kryptofix 2.2.2. RESULTS: After purification on solid-phase extraction cartridges, the [18F]fluorocholine was obtained in 15-25% radiochemical yields (decay not corrected), with more than 99% radiochemical purity. Specific activity was more than 37 GBq/micromol. Synthesis time was less than 35 min. CONCLUSION: This new automated synthesis technique provides high and reproducible yields that could be dedicated for routine use with the same [18F]FDG disposable cassette system.     

Fully automated synthesis of PET TSPO radioligands [11C]DAA1106 and [18F]FEDAA1106

[(11)C]DAA1106 was prepared by O-[(11)C]methylation of DAA1123 with [(11)C]CH(3)OTf and NaH in CH(3)CN at 80°C and isolated by HPLC combined with SPE purification in 60-70% decay corrected radiochemical yield. [(18)F]FEDAA1106 was synthesized by the nucleophilic substitution of tosyloxy-FEDAA1106 in DMSO with K[(18)F]F/Kryptofix 2.2.2 at 140°C and isolated by HPLC combined with SPE purification in 30-60% decay corrected radiochemical yield. The specific activity for [(11)C]DAA1106 and [(18)F]FEDAA1106 was 370-740GBq/μmol and 37-222GBq/μmol at EOB, respectively.     

Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction

Introduction4-[¹⁸F]Fluorobenzylamine ([¹⁸F]FBA) is an important building block for the synthesis of ¹⁸F-labeled compounds. Synthesis of [¹⁸F]FBA usually involves application of strong reducing agents like LiAlH₄ which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [¹⁸F]FBA compatible with remotely-controlled syntheses in ASUs are needed.Methods¹⁸F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab? FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[¹⁸F]fluorobenzonitrile ([¹⁸F]FBN). [¹⁸F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[¹⁸F]fluorobenzyl)maleimide [¹⁸F]FBM and Hsp90 inhibitor 17-(4-[¹⁸F]fluorobenzylamino)-17-demethoxy-geldanamycin [¹⁸F] GA.Results[¹⁸F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60 min. In a typical experiment, 7.4 GBq of [¹⁸F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10 GBq of cyclotron-produced n.c.a. [¹⁸F]fluoride. [¹⁸F]FBA was used for the preparation of 4-[¹⁸F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [¹⁸F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[¹⁸F]fluorobenzylamino)-17-demethoxy-geldanamycin.ConclusionThe described remotely-controlled synthesis of [¹⁸F]FBA will significantly improve the availability of [¹⁸F]FBA as an important and versatile building block for the development of novel ¹⁸F-labeled compounds containing a fluorobenzylamine moiety.     

NanoPET imaging of [18F]fluoromisonidazole uptake in experimental mouse tumours

Purpose The purpose of this study was to assess the potential and utility of ultra-high-resolution hypoxia imaging in various murine tumour models using the established hypoxia PET tracer [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO).Methods [¹⁸F]FMISO PET imaging was performed with the dedicated small-animal PET scanner NanoPET (Oxford Positron Systems) and ten different human tumour xenografts in nude mice as well as B16 melanoma tumours in syngeneic Balb/c mice. For comparison, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) PET scans were also performed in the mice bearing human tumour xenografts.Results In 10 out of 11 experimental tumour models, [¹⁸F]FMISO PET imaging allowed clear-cut visualisation of the tumours. Inter- and intratumoural heterogeneity of tracer uptake was evident. In addition to average TMRR (tumour-to-muscle retention ratio including all voxels in a volume of interest (VOI)), the parameters TMRR_75% and TMRR₅ (tumour-to-muscle retention ratio including voxels of 75% or more of the maximum radioactivity in a VOI and the five hottest pixels, respectively) also served as measures for quantifying the heterogeneous [¹⁸F]FMISO uptake in the tumours. The variability observed in [¹⁸F]FMISO uptake was related neither to tumour size nor to the injected mass of the radiotracer. The pattern of normoxic and hypoxic regions within the human tumour xenografts, however, correlated with glucose metabolism as revealed by comparison of [¹⁸F]FDG and [¹⁸F]FMISO images.Conclusion This study demonstrates the feasibility and utility of [¹⁸F]FMISO for imaging murine tumour models using NanoPET.     

The automated radiosynthesis of [18F]FP-TZTP

[(18)F]FP-TZTP (3-(3-(3-[(18)F]fluoropropylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine) is a muscarinic ligand that displays in vivo selectivity for the M2 subtype. We have developed a one-step radiosynthesis of [(18)F]FP-TZTP that can be conducted with an automated synthesis unit. A number of hardware and software modifications to a Nuclear Interface C-11 Methylation System provided the equipment for the automated radiosynthesis. The manual synthesis produced [(18)F]FP-TZTP in a radiochemical yield of 23.4% +/- 4.3% (EOS, n = 69) with a specific activity of 4377 +/- 2011 mCi/micromol (EOB, n = 100). The automated synthesis unit provided the product in a radiochemical yield of 18.8% +/- 2.4% (EOS, n = 25) with a specific activity of 4112 +/- 2572 mCi/micromol (EOB, n = 25).     

Improved synthesis of [18F]FLETT via a fully automated vacuum distillation method for [18F]2-fluoroethyl azide purification

The synthesis of [¹⁸F]2-fluoroethyl azide and its subsequent click reaction with 5-ethynyl-2′-deoxyuridine (EDU) to form [¹⁸F]FLETT was performed using an iPhase FlexLab module. The implementation of a vacuum distillation method afforded [¹⁸F]2-fluoroethyl azide in 87±5.3% radiochemical yield. The use of Cu(CH₃CN)₄PF₆ and TBTA as catalyst enabled us to fully automate the [¹⁸F]FLETT synthesis without the need for the operator to enter the radiation field. [¹⁸F]FLETT was produced in higher overall yield (41.3±6.5%) and shorter synthesis time (67 min) than with our previously reported manual method (32.5±2.5% in 130 min).     

Fully automated synthesis procedure of 4-[18F]fluorobenzaldehyde by commercial synthesizer: Amino-oxi peptide labelling prosthetic group

Automatic synthesis of 4-[18F]fluorobenzaldehyde has been developed by a commercially available TRACERlab™ FX_F−N synthesis module to be used as prosthetic group for amino-oxy functionalized peptide labelling in clinical routine application. In addition a handmade purification device (HPD) has been setup to perform automatic cartridge purification as well as to back-up the reactor where one-pot synthesis is not applicable. Cartridges for solid phase extraction such as C18, C8, phenyl has been tested to best perform purification as well as activity recovery. Radiochemical yield (RCY) at end of synthesis (EOS) was in average 67% after about 45 min (90% decay corrected at EOB). The RCY of the entire procedure was 54% with a radiochemical purity above 99%.     

The synthesis of [18F]Xenon difluoride from [18F]fluoride gas

A convenient procedure for the rapid synthesis of ¹⁸F labelled XeF₂ was developed. Neon gas with 0.087 to 5% carrier fluorine was irradiated with 15 MeV deuterons to produce [¹⁸F]F₂ with a yield of 17 mCi/μAh, EOB. It was, in turn, reacted with excess of xenon in a high pressure nickel vessel at 390°C for 40 min. The isolated reaction product was XeF₂ exclusively. The production yield was 11 mCi/μAh at EOB and the specific activity was 450mCi/mmol. [¹⁸F]XeF₂ has been shown to be a versatile intermediate for the syntheses of ¹⁸F-labelled radiopharmaceuticals, for example, [¹⁸F]2-fluoro-2-deoxy-D-glucose and L-[¹⁸F]6-fluorodopa.     

An automated one-step one-pot [(18)F]FCWAY synthesis: development and minimization of chemical impurities

[(18)F]FCWAY (N-{2-[4-(2-methoxyphenyl)piperazino]}-N-(2-pyridinyl)trans-4-fluorocyclohexanecarboxamide) has been prepared routinely as a serotonin 5-HT(1A) receptor ligand for clinical human studies. We have developed an automated one-step radiosynthesis using a modified Nuclear Interface C-11 Methylation System. The chemical synthesis of an appropriate methanesulfonate precursor for single-step nucleophilic substitution with [(18)F]fluoride ion and the adaptation of radiochemical synthesis to an automated production module were accomplished. Following purification of a substrate using countercurrent chromatography, radiochemical yield increased from 18.9+/-0.3% to 21.9+/-2.2%. In addition, reduction of chemical impurities from about 40% to about 20% of total mass was observed. Further improvements in chemical purity were achieved by minimization of side reactions by modification of reaction conditions and optimization of the high-performance liquid chromatography method for the purification of the final radiopharmaceutical. Optimized automated synthesis produced [(18)F]FCWAY in a radiochemical yield of 28+/-6% at a chemical purity of 99.3% based on the absorbance of FCWAY at 254 nm and with a specific activity of 3433+/-1015 mCi/micromol at the end of bombardment, all calculated from the same 50 runs.     

Radiochemical synthesis of [18F]fluororaclopride

The radiochemical synthesis of [¹⁸F]Fluororaclopride (S-3,5-dichloro-6-methoxy-N-(1-(2-[¹⁸F]fluoro-ethyl)-2-pyrrolidinylmethyl)salicylamide) is accomplished via a two step synthesis. [¹⁸F]Fluoroethyltriflate is prepared by [¹⁸F]fluoride displacement on the bis triflate of ethylene glycol. [¹⁸F]Fluoroethyl triflate is then allowed to alkylate a secondary amine precursor to yield [¹⁸F]fluororaclopride. Purification of the radiopharmaceutical involves use of a short silica BONDELUT column and subsequent reverse-phase HPLC. The final product is obtained with a radiochemical yield of approximately 15% (corrected for decay) in a synthesis time of approximately 50 min. Fluororaclopride displays a 3- to 4-fold lower affinity for the D₂ receptor than does raclopride.     

Fully automated radiosynthesis of both enantiomers of [18F]Flubatine under GMP conditions for human application

A fully automatized radiosynthesis of (+)- and (−)-[¹⁸F]Flubatine ((+)- and (−)NCFHEB) by means of a commercially available synthesis module (TRACERlab FX FN) under GMP conditions is reported. Radiochemical yields of 30% within an overall synthesis time of 40 min were achieved in more than 70 individual syntheses. Specific activities were approximately 3000 GBq/μmol and radiochemical purity was determined to be at least 97%.     

Regioselective radiofluorodestannylation with [18F]F2 and [18F]CH3COOF: A high yield synthesis of 6-[18F]fluoro-l-dopa

A protected 6-trimethylstannyl dopa derivative 6 has been synthesized for the first time as a precursor for the preparation of 6-[¹⁸F]fluoro-l-dopa. The tin derivative 6 readily reacted with electrophilic radiofluorinating agents such as [¹⁸F]F₂ and [¹⁸F]AcOF. The [¹⁸F]fluoro intermediate 7 was easily hydrolyzed with HBr and the product 6-[¹⁸F]fluoro-l-dopa was isolated after HPLC purification in a maximum radiochemical yield of 25%, ready for human use. The various intermediates, the stannyl precursor 6 and the final product (after ¹⁸F decay) were all fully characterized by ¹H, ¹³C, ¹⁹F and ¹¹⁹Sn NMR as well as high resolution mass spectroscopy.     

A “dose on demand” Biomarker Generator for automated production of [18F]F− and [18F]FDG

The University of Oklahoma—College of Pharmacy has installed the first Biomarker Generator (BG75) comprising a self-shielded 7.5-MeV proton beam positive ion cyclotron and an aseptic automated chemistry production and quality control module for production of [¹⁸F]F⁻ and clinical [¹⁸F]FDG. Performance, reliability, and safety of the system for the production of “dose on demand” were tested over several months. No-carrier-added [¹⁸F]F⁻ was obtained through the ¹⁸O(p,n)¹⁸F nuclear reaction by irradiation (20–40 min) of a >95% enriched [¹⁸O]H₂O target (280 μl) with a 7.5-MeV proton beam (3.5–5.0 μA). Automated quality control tests were performed on each dose. The HPLC-based analytical methods were validated against USP methods of quality control. [¹⁸F]FDG produced by BG75 was tested in a mouse tumor model implanted with H441 human lung adenocarcinoma cells. After initial installment and optimization, the [¹⁸F]F⁻ production has been consistent since March 2011 with a maximum production of 400 to 450 mCi in a day. The average yield is 0.61 mCi/min and 0.92 mCi/min at 3.8 µA and 5 µA, respectively. The current target window has held up for over 25 weeks against >400 bombardment cycles. [¹⁸F]FDG production has been consistent since June 2012 with an average of six doses/day in an automated synthesis mode (RCY≈50%). The release criteria included USP-specified limits for pH, residual solvents (acetonitrile/ethanol), kryptofix, radiochemical purity/identity, and filter integrity test. The entire automated operation generated minimal radiation exposure hazard to the operator and environment. As expected, [¹⁸F]FDG produced by BG75 was found to delineate tumor volume in a mouse model of xenograft tumor. In summary, production and quality control of “[¹⁸F]FDG dose on demand” have been accomplished in an automated and safe manner by the first Biomarker Generator. The implementation of a cGMP quality system is under way towards the ANDA submission and first clinical use of [¹⁸F]FDG produced by BG75.     

Development of microwave-based automated nucleophilic [18F]fluorination system and its application to the production of [18F]flumazenil

IntroductionThis study presents the development of an automated radiosynthesis system integrating a microwave reactor and its subsequent application in the synthesis of [¹⁸F]flumazenil, a potentially useful compound in the evaluation of central benzodiazepine receptor density.MethodsPreparation of dry [K/K₂₂₂]⁺¹⁸F^? complex and radiofluorination of the nitro-flumazenil precursor were achieved using the developed microwave-based radiosynthesis system. The crude product was prepurified in a C18 cartridge followed by reversed-phase preparative high-performance liquid chromatography. The isolated [¹⁸F]flumazenil was evaporated in vacuo and reconstituted in an ethanol-free solution.ResultsOptimum incorporation of ¹⁸F^? in the nitro-precursor was achieved in 5 min time utilizing 2 mg of precursor in N,N-dimethylformamide reacted at 160°C which gave an incorporation yield of 40±5%. The radiochemical yield obtained at the end of synthesis was 26±4% (EOB) with a radiochemical purity of >99% and a total synthesis time of about 55–60 min. The produced [¹⁸F]flumazenil was observed to be stable for at least 8 h.ConclusionThe developed [¹⁸F]flumazenil radiosynthesis system offers shorter reaction time, simplicity in operation and applicability for use in routine clinical practice.