Giancarlo Pascali

Microfluidic approach for fast labeling optimization and dose-on-demand implementation

INTRODUCTION The diffusion of PET as a pivotal molecular imaging modality has emphasized the need for new positron-emitting radiotracers to be used in diagnostic applications and research. Microfluidic represents an innovative approach, owing to its potential to increase radiochemical productivity in terms of yields, time reduction, precursor consumption and flexible experimental planning. METHODS We focused on fluorine-18 labeling and used a microfluidic platform to perform sequential reactions, by using the same batch of (18)F-labeling solution on one or more substrates, during the same experimental session. A solid-phase extraction (SPE) workup procedure was also implemented in the system to provide a repeatable purification step. RESULTS We were able to quickly optimize the conditions for labeling of ethyl and propyl ditosylate and of a new cannabinoid type 2 (CB2) receptor agonist, CB41. In all substrates, we obtained good incorporation yields (60% to 85%) in short (<90 s) reaction times. Single dosages of the CB2 ligand were sequentially prepared, upon request, in satisfactory quantities and purity for small animal PET scanning. CONCLUSION This work demonstrates the usefulness of a microfluidic-based system for a rapid optimization of temperature, flow rate of reactants and their relative ratio in the labeling of different precursors by using the same (18)F-fluoride batch. This approach was used to obtain in sequence several injectable doses of a novel CB2 ligand, thus providing the proof of principle that microfluidic systems permit a dose-on-demand production of new radiotracers.

Microfluidics in radiopharmaceutical chemistry

The increased demand for molecular imaging tracers useful in assessing and monitoring diseases has stimulated research towards more efficient and flexible radiosynthetic routes, including newer technologies. The traditional vessel-based approach suffers from limitations concerning flexibility, reagent mass needed, hardware requirements, large number of connections and valves, repetitive cleaning procedures and overall big footprint to be shielded from radiation. For these reasons, several research groups have started to investigate the application of the fast growing field of microfluidic chemistry to radiosynthetic procedures. After the first report in 2004, many scientific papers have been published and demonstrated the potential for increased process yields, reduced reagent use, improved flexibility and general ease of setup. This review will address definitions occurring in microfluidics as well as analyze the different approaches under two macro-categories: microvessel and microchannel. In this perspective, several works will be collected, involving the use of positron emitting species ((11)C, (18)F, (64)Cu) and the fewer examples of gamma emitting radionuclides ((99m)Tc, (125/131)I). New directions in microfluidic research applied to PET radiochemistry, future developments and challenges are also discussed.

Dose-on-demand of diverse 18F-fluorocholine derivatives through a two-step microfluidic approach

INTRODUCTION The validation and confirmation of clinical usefulness of new and known positron emission tomography (PET) tracers require stable production routes and simple and robust radiochemical procedures. Microfluidic technologies are regarded as an approach that could allow an unprecedented flexibility and productivity in PET radiopharmaceutical research. In this work, we will show how a commercially available microfluidic system can be used for a sequential and repeatable radiosynthesis of three different fluorocholine analogues currently under investigation as tumor tracers. METHODS Advion microfluidic system was used for performing the synthesis and purification of [(18)F]fluoromethyl, [(18)F]fluoroethyl or [(18)F]fluoropropyl choline employing a two-step approach, starting from the corresponding alkyl-ditosylate and reacting the [(18)F]fluorotosylate obtained in the first step with neat dimethylethanolamine. The purification was obtained using a recyclable SPE cartridge set. RESULTS The three products, fluoromethylcholine, fluoroethylcholine and fluoropropylcholine, were obtained in good to optimum yields (22%-54% decay corrected) with a 15-min procedure. The production could be restarted several times for producing each one of the tracers without decrease in yields and purities, in accordance with a dose-on-demand (DOD) approach. The final products were formulated in isotonic saline solution. CONCLUSION The described approach gives a proof of principle of the enhanced productivity obtainable using a microfluidic approach; in particular, the possibility to produce the reported tracers in a DOD fashion following a homogeneous synthetic and purification approach will foster further studies on the clinical evaluation of the best fluorocholine analogue for prostate cancer imaging without biasing for differences in radiochemical approach.

Radiochemistry on chip

We have developed an integrated microfluidic platform for producing 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) in continuous flow from a single bolus of radioactive isotope solution, with constant product yields achieved throughout the operation that were comparable to those reported for commercially available vessel-based synthesisers (40-80%). The system would allow researchers to obtain radiopharmaceuticals in a dose-on-demand setting within a few minutes. The flexible architecture of the platform, based on a modular design, can potentially be applied to the synthesis of other radiotracers that require a two-step synthetic approach, and may be adaptable to more complex synthetic routes by implementing additional modules. It can therefore be employed for standard synthesis protocols as well as for research and development of new radiopharmaceuticals.

On-chip pre-concentration and complexation of [18F]fluoride ions via regenerable anion exchange particles for radiochemical synthesis of Positron Emission Tomography tracers

Microfluidic approaches have demonstrated a relevant impact on radiochemical reactions involving Positron Emission Tomography (PET) nuclides, due to shorter reaction times and smaller precursor quantities. However, little attention has been given to the integration of the initial pre-concentration and drying of radioactive [(18)F]fluoride ions, required for the labeling of radiotracer compounds. In this work we report the design, fabrication and implementation of a glass microfluidic device filled with recyclable anion exchange particles for the repeated recovery of [(18)F] and [(19)F]fluoride ions. The device was first tested with non radioactive [(19)F]fluoride ions and it was shown to repeatedly trap and elute >95% fluoride over 40 successive experimental runs with no decrease in efficiency. The same device was then tested for the trapping and release of [(18)F]fluoride ions over 20 experiments with no measurable decrease in performance. Finally, the [(18)F]fluoride ions were eluted as a K(18)F/K2.2.2 complex, dried by repeated dissolution in acetonitrile and evaporation of residual water, and reacted with ethyl ditosylate (EtDT) leading to the desired product ([(18)F]fluoroethyltosylate) with 96 ± 3% yield (RCY). The overall time needed for conditioning, trapping, elution and regeneration was less than 6 min. This approach will be of great benefit towards an integrated platform able to perform faster and safer radiochemical synthesis on the micro-scale.

Positron Emission Tomography Radiosynthesis in Microreactors

Positron emission tomography (PET) is a very powerful diagnostic technique routinely used in a variety of medical applications. This review article summarises the developments of using microreactor technology for the radiochemical synthesis of PET agents. The advantages of manufacturing these imaging drugs using microreactors are described, and a review of reactions conducted to date is outlined.

Optimization of nucleophilic 18F radiofluorinations using a microfluidic reaction approach

Microfluidic techniques are increasingly being used to synthesize positron-emitting radiopharmaceuticals. Several reports demonstrate higher incorporation yields, with shorter reaction times and reduced amounts of reagents compared with traditional vessel-based techniques. Microfluidic techniques, therefore, have tremendous potential for allowing rapid and cost-effective optimization of new radiotracers. This protocol describes the implementation of a suitable microfluidic process to optimize classical 18F radiofluorination reactions by rationalizing the time and reagents used. Reaction optimization varies depending on the systems used, and it typically involves 5–10 experimental days of up to 4 h of sample collection and analysis. In particular, the protocol allows optimization of the key fluidic parameters in the first tier of experiments: reaction temperature, residence time and reagent ratio. Other parameters, such as solvent, activating agent and precursor concentration need to be stated before the experimental runs. Once the optimal set of parameters is found, repeatability and scalability are also tested in the second tier of experiments. This protocol allows the standardization of a microfluidic methodology that could be applied in any radiochemistry laboratory, in order to enable rapid and efficient radiosynthesis of new and existing [18F]-radiotracers. Here we show how this method can be applied to the radiofluorination optimization of [18F]-MEL050, a melanoma tumor imaging agent. This approach, if integrated into a good manufacturing practice (GMP) framework, could result in the reduction of materials and the time required to bring new radiotracers toward preclinical and clinical applications.

Optimization of automated large-scale production of [18F] fluoroethylcholine for PET prostate cancer imaging

INTRODUCTION PET tumor imaging is gaining importance in current clinical practice. FDG-PET is the most utilized approach but suffers from inflammation influences and is not utilizable in prostate cancer detection. Recently, (11)C-choline analogues have been employed successfully in this field of imaging, leading to a growing interest in the utilization of (18)F-labeled analogues: [(18)F]fluoroethylcholine (FEC) has been demonstrated to be promising, especially in prostate cancer imaging. In this work we report an automatic radiosynthesis of this tracer with high yields, short synthesis time and ease of performance, potentially utilizable in routine production sites. METHODS We used a Modular Lab system to automatically perform the two-step/one-pot synthesis. In the first step, we labeled ethyleneglycolditosylate obtaining [(18)F]fluoroethyltosylate; in the second step, we performed the coupling of the latter intermediate with neat dimethylethanolamine. The final mixture was purified by means of solid phase extraction; in particular, the product was trapped into a cation-exchange resin and eluted with isotonic saline. RESULTS The optimized procedure resulted in a non decay corrected yield of 36% and produced a range of 30-45 GBq of product already in injectable form. The product was analyzed for quality control and resulted as pure and sterile; in addition, residual solvents were under the required threshold. CONCLUSION In this work, we present an automatic FEC radiosynthesis that has been optimized for routine production. This findings should foster the interest for a wider utilization of this radiomolecule for imaging of prostate cancer with PET, a field for which no gold-standard tracer has yet been validated.

Design, synthesis and preliminary evaluation of 18F-labelled 1,8-naphthyridin- and quinolin-2-one-3-carboxamide derivatives for PET imaging of CB2 cannabinoid receptor

In the present work, we report the synthesis of new aryliodonium salts used as precursors of single-stage nucleophilic (18)F radiofluorination. The corresponding unlabelled fluorinated derivatives showed to be CB2 cannabinoid receptor specific ligands, with Ki values in the low nanomolar range and high CB2/CB1 selectivity. The radiolabelled compound [(18)F]CB91, was successfully formulated for in vivo administration, and its preliminary biodistribution was assessed with microPET/CT. This tracer presented a reasonable in vivo stability and a preferential extraction in the tissues that constitutionally express CB2 cannabinoid receptor. The results obtained indicate [(18)F]CB91 as a possible candidate marker of CB2 cannabinoid receptor distribution. This study would open the way to further validation of this tracer for assessing pathologies for which the expression of this receptor is modified.

Purification of 2-[18F]fluoro-2-deoxy-d-glucose by on-chip solid-phase extraction.

Microfluidic devices have shown great potential for the production of positron emission tomography (PET) radiotracers, but most devices have focused only on the synthesis step of the procedure, typically neglecting the other important steps such as [(18)F]fluoride pre-concentration and radiotracer purification that could equally benefit from miniaturisation. Here, we demonstrate the development of microfluidic modules for the purification of PET radiotracers, particularly 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG), via the use of on-chip solid-phase extraction (SPE). In these initial tests, the SPE modules were able to yield [(18)F]FDG with up to 90% radiochemical purity, and methods are proposed for further increasing this value.