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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2021)

Positron emission tomography (PET) is a powerful medical diagnostics and research tool that uses radiolabeled molecules (tracers) to image biological processes in vivo. By administering only nanomolar quantities of the tracers, PET scans enable non-invasive assessment of normal biological processes in cells and their failure in disease to aid in medical diagnostics, staging of disease severity and monitoring treatment response. Short-lived radioisotopes used in the synthesis of diagnostic PET tracers necessitate that the tracer production is carried out shortly before imaging. Each production is a multistep process involving acquisition of the radioisotope, radiochemical synthesis and quality control. Radiochemical synthesis is further broken down into radiochemical reaction to link the radioisotope with a ligand, purification and formulation to obtain pure injection-ready product.Despite impressive sensitivity and accuracy of PET in medical diagnostics, access to the wide variety of short-lived radioactive tracers is hindered due to a high cost of their preparation. The overall preparation cost covers: (i) radiosynthesis units and various consumables, (ii) large shielded fume hoods (hot-cells), (iii) reagents and radioisotope, (iv) labor and safety. A typical centralized production of PET tracers in large radiochemistry facilities alleviates the high cost per production by splitting of the large batches and shipping those to multiple users, which is only possible for tracers in high demand such as [18F]fluorodeoxyglucose ([18F]FDG). Newly introduced dose-on-demand concept proposes convenient production of any tracer of interest directly at the imaging location, leading to a decentralized approach. While setting up dedicated conventional radiosynthesis modules for this purpose is unrealistic due to the high cost and large footprint, microfluidic approaches provide a path for a miniaturized, economical tracer production. Our lab among others has been extensively working on the radiosynthesis miniaturization using droplet microfluidic methods. Microfluidics offers potential of cost reduction in almost all aspects of radiosynthesis. (i) The synthesizer cost can be reduced as a result of reduced system size, complexity and consumables cost, (ii) the compact units can be self-shielded not requiring use of hot-cells, (iii) the reagent and radioisotope consumption per synthesis is orders of magnitude less, (iv) the faster synthesis times and small-scale production levels reduce labor burden and improve safety. Additionally, in fluorine-18 radiochemistry, the conventional synthesizers must use high starting radioactivity to ensure good molar activity (radioactivity per moles of the substance) of the final product. Previously, our group has developed automated droplet reactors based on electrowetting on dielectric (EWOD) technology. However, those devices had limitations due to high cost and complexity of fabrication. More recently, we introduced low-cost disposable silicon chips which serve as a reactor within an ultra-compact automated radiosynthesizer unit, comparable in size to a 12 oz. coffee cup. The major part of this dissertation was adapting various conventional fluorine-18-labeling synthesis methods to a droplet format to demonstrate the versatility of this radiosynthesis approach. For a number of tracers, we describe synthesis miniaturization process and demonstrate that the droplet syntheses exhibit higher yields and improved synthesis times in comparison to the conventional methods. All syntheses feature orders-of-magnitude reduction in reagent consumption and are able to achieve high molar activity even when lower starting amounts of the radioisotope are used. As a result, low cost per production can be achieved and such methods are readily useable for research in particular that involves small animal PET imaging. Importantly, we further demonstrate that these syntheses are scalable, and that production of a few human doses is feasible. For validation, we also perform full clinical quality control on these tracer batches. These results suggest that in the future it would be possible to introduce this technology in a clinical setting as well for an easy access to a wide variety of PET tracers. From the radiochemistry standpoint, microfluidic fluorine-18 radiolabeling has been shown to work for different type of ligands. Apart from small molecule radiolabeling, peptide labeling routes via droplet radiochemistry are also shown. In this work we feature isotopic exchange (IEX) fluorination that has a benefit of simplified purification but is challenging on conventional scale due to inherently lower molar activity in this type of reactions. We demonstrate that the droplet approach with its small volumes allows one to perform IEX synthesis of trifluoroborate-based peptides and prosthetic groups with high yields and molar activities. This dissertation presents reliable methods for fluorine-18-labeled radiopharmaceutical production on demand, in a time- and cost-efficient manner for diverse PET tracers. While with the current progress, these methods can be readily applied for research purposes including pre-clinical PET imaging, and future work will focus on improvements and optimization of the droplet microfluidic technologies to advance these methods to the clinical PET.

    Cover page: Novel economical approaches for the fluorine-18 radiopharmaceuticals production via droplet radiochemistry
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2020)
    From an efficiency standpoint, microdroplet reactors enable significant improvements in the preparation of radiopharmaceuticals due to the vastly reduced reaction volume. To demonstrate these advantages, we adapt the conventional (macroscale) synthesis of the clinically-important positron-emission tomography tracer [18F]FDOPA, following the nucleophilic diaryliodonium salt approach, to a newly-developed ultra-compact microdroplet reaction platform. In this first microfluidic implementation of [18F]FDOPA synthesis, optimized via a high-throughput multi-reaction platform, the radiochemical yield (non-decay-corrected) was found to be comparable to macroscale reports, but the synthesis consumed significantly less precursor and organic solvents, and the synthesis process was much faster. In this initial report, we demonstrate the production of [18F]FDOPA in 15 MBq [400 μCi] amounts, sufficient for imaging of multiple mice, at high molar activity.
      Cover page: Green and efficient synthesis of the radiopharmaceutical [ 18 F]FDOPA using a microdroplet reactor
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)

      Background

      Current automated radiosynthesizers are generally optimized for producing large batches of PET tracers. Preclinical imaging studies, however, often require only a small portion of a regular batch, which cannot be economically produced on a conventional synthesizer. Alternative approaches are desired to produce small to moderate batches to reduce cost and the amount of reagents and radioisotope needed to produce PET tracers with high molar activity. In this work we describe the first reported microvolume method for production of [18F]Florbetaben for use in imaging of Alzheimer's disease.

      Procedures

      The microscale synthesis of [18F]Florbetaben was adapted from conventional-scale synthesis methods. Aqueous [18F]fluoride was azeotropically dried with K2CO3/K222 (275/383 nmol) complex prior to radiofluorination of the Boc-protected precursor (80 nmol) in 10 μL DMSO at 130 °C for 5 min. The resulting intermediate was deprotected with HCl at 90 °C for 3 min and recovered from the chip in aqueous acetonitrile solution. The crude product was purified via analytical scale HPLC and the collected fraction reformulated via solid-phase extraction using a miniature C18 cartridge.

      Results

      Starting with 270 ± 100 MBq (n = 3) of [18F]Fluoride, the method affords formulated product with 49 ± 3% (decay-corrected) yield,> 98% radiochemical purity and a molar activity of 338 ± 55 GBq/μmol. The miniature C18 cartridge enables efficient elution with only 150 μL of ethanol which is diluted to a final volume of 1.0 mL, thus providing a sufficient concentration for in vivo imaging. The whole procedure can be completed in 55 min.

      Conclusions

      This work describes an efficient and reliable procedure to produce [18F]Florbetaben in quantities sufficient for large-scale preclinical applications. This method provides very high yields and molar activities compared to reported literature methods. This method can be applied to higher starting activities with special consideration given to automation and radiolysis prevention.
        Cover page: A simple and efficient automated microvolume radiosynthesis of [18F]Florbetaben
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2020)

        Introduction

        Radio thin layer chromatography (radio-TLC) is commonly used to analyze purity of radiopharmaceuticals or to determine the reaction conversion when optimizing radiosynthesis processes. In applications where there are few radioactive species, radio-TLC is preferred over radio-high-performance liquid chromatography due to its simplicity and relatively quick analysis time. However, with current radio-TLC methods, it remains cumbersome to analyze a large number of samples during reaction optimization. In a couple of studies, Cerenkov luminescence imaging (CLI) has been used for reading radio-TLC plates spotted with a variety of isotopes. We show that this approach can be extended to develop a high-throughput approach for radio-TLC analysis of many samples.

        Methods

        The high-throughput radio-TLC analysis was carried out by performing parallel development of multiple radioactive samples spotted on a single TLC plate, followed by simultaneous readout of the separated samples using Cerenkov imaging. Using custom-written MATLAB software, images were processed and regions of interest (ROIs) were drawn to enclose the radioactive regions/spots. For each sample, the proportion of integrated signal in each ROI was computed. Various crude samples of [18F]fallypride, [18F]FET and [177Lu]Lu-PSMA-617 were prepared for demonstration of this new method.

        Results

        Benefiting from a parallel developing process and high resolution of CLI-based readout, total analysis time for eight [18F]fallypride samples was 7.5 min (2.5 min for parallel developing, 5 min for parallel readout), which was significantly shorter than the 48 min needed using conventional approaches (24 min for sequential developing, 24 min for sequential readout on a radio-TLC scanner). The greater separation resolution of CLI enabled the discovery of a low-abundance side product from a crude [18F]FET sample that was not discernable using the radio-TLC scanner. Using the CLI-based readout method, we also observed that high labeling efficiency (99%) of [177Lu]Lu-PSMA-617 can be achieved in just 10 min, rather than the typical 30 min timeframe used.

        Conclusions

        Cerenkov imaging in combination with parallel developing of multiple samples on a single TLC plate proved to be a practical method for rapid, high-throughput radio-TLC analysis.
          Cover page: High-throughput radio-TLC analysis
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2021)
          Current equipment and methods for preparation of radiopharmaceuticals for positron emission tomography (PET) are expensive and best suited for large-scale multi-doses batches. Microfluidic radiosynthesizers have been shown to provide an economic approach to synthesize these compounds in smaller quantities, but can also be scaled to clinically-relevant levels. Batch microfluidic approaches, in particular, offer significant reduction in system size and reagent consumption. Here we show a simple and rapid technique to concentrate the radioisotope, prior to synthesis in a droplet-based radiosynthesizer, enabling production of clinically-relevant batches of [18F]FET and [18F]FBB. The synthesis was carried out with an automated synthesizer platform based on a disposable Teflon-silicon surface-tension trap chip. Up to 0.1 mL (4 GBq) of radioactivity was used per synthesis by drying cyclotron-produced aqueous [18F]fluoride in small increments directly inside the reaction site. Precursor solution (10 µL) was added to the dried [18F]fluoride, the reaction chip was heated for 5 min to perform radiofluorination, and then a deprotection step was performed with addition of acid solution and heating. The product was recovered in 80 µL volume and transferred to analytical HPLC for purification. Purified product was formulated via evaporation and resuspension or a micro-SPE formulation system. Quality control testing was performed on 3 sequential batches of each tracer. The method afforded production of up to 0.8 GBq of [18F]FET and [18F]FBB. Each production was completed within an hour. All batches passed quality control testing, confirming suitability for human use. In summary, we present a simple and efficient synthesis of clinically-relevant batches of [18F]FET and [18F]FBB using a microfluidic radiosynthesizer. This work demonstrates that the droplet-based micro-radiosynthesizer has a potential for batch-on-demand synthesis of 18F-labeled radiopharmaceuticals for human use.
            Cover page: Economical droplet-based microfluidic production of [18F]FET and [18F]Florbetaben suitable for human use
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2020)

            Background

            Conventional scale production of small batches of PET tracers (e.g. for preclinical imaging) is an inefficient use of resources. Using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET), we demonstrate that simple microvolume radiosynthesis techniques can improve the efficiency of production by consuming tiny amounts of precursor, and maintaining high molar activity of the tracers even with low starting activity.

            Procedures

            The synthesis was carried out in microvolume droplets manipulated on a disposable patterned silicon "chip" affixed to a heater. A droplet of [18F]fluoride containing TBAHCO3 was first deposited onto a chip and dried at 100 °C. Subsequently, a droplet containing 60 nmol of precursor was added to the chip and the fluorination reaction was performed at 90 °C for 5 min. Removal of protecting groups was accomplished with a droplet of HCl heated at 90 °C for 3 min. Finally, the crude product was collected in a methanol-water mixture, purified via analytical-scale radio-HPLC and formulated in saline. As a demonstration, using [18F]FET produced on the chip, we prepared aliquots with different molar activities to explore the impact on preclinical PET imaging of tumor-bearing mice.

            Results

            The microdroplet synthesis exhibited an overall decay-corrected radiochemical yield of 55 ± 7% (n = 4) after purification and formulation. When automated, the synthesis could be completed in 35 min. Starting with < 370 MBq of activity, ~ 150 MBq of [18F]FET could be produced, sufficient for multiple in vivo experiments, with high molar activities (48-119 GBq/μmol). The demonstration imaging study revealed the uptake of [18F]FET in subcutaneous tumors, but no significant differences in tumor uptake as a result of molar activity differences (ranging 0.37-48 GBq/μmol) were observed.

            Conclusions

            A microdroplet synthesis of [18F]FET was developed demonstrating low reagent consumption, high yield, and high molar activity. The approach can be expanded to tracers other than [18F]FET, and adapted to produce higher quantities of the tracer sufficient for clinical PET imaging.
              Cover page: Rapid, efficient, and economical synthesis of PET tracers in a droplet microreactor: application to O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET)
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2018)
              The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293 cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [18F]4-N-alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale and conventional silane-labeling radiochemical protocols. Preliminary positron-emission tomography (PET) imaging in mice showed the biodistribution of [18F]4-N-alkyl to have initial concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.
                Cover page: The 4-N-acyl and 4-N-alkyl gemcitabine analogues with silicon-fluoride-acceptor: Application to 18F-Radiolabeling
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)

                Background

                Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced.

                Methods

                Both [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE.

                Results

                The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50 ± 6% (n = 15), overall decay-corrected radiochemical yield of 16 ± 1% (n = 5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26 × 10-2 mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds.

                Conclusions

                The combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.
                  Cover page: Microscale radiosynthesis, preclinical imaging and dosimetry study of [18F]AMBF3-TATE: A potential PET tracer for clinical imaging of somatostatin receptors
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