First Human Use of a Radiopharmaceutical Prepared by Continuous-Flow Microfluidic Radiofluorination: Proof of Concept with the Tau Imaging Agent [ 18 F]T807

Abstract

Despite extensive preclinical imaging with radiotracers developed by continuous-flow microfluidics, a positron emission tomographic (PET) radiopharmaceutical has not been reported for human imaging studies by this technology. The goal of this study was to validate the synthesis of the tau radiopharmaceutical 7-(6-fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([¹⁸F]T807) and perform first-in-human PET scanning enabled by microfluidic flow chemistry. [¹⁸F]T807 was synthesized by our modified one-step method and adapted to suit a commercial microfluidic flow chemistry module. For this proof of concept, the flow system was integrated to a GE Tracerlab FX_FN unit for high-performance liquid chromatography purification and formulation. Three consecutive productions of [¹⁸F]T807 were conducted to validate this radiopharmaceutical. Uncorrected radiochemical yields of 17 ± 1% of crude [¹⁸F]T807 (≈ 500 mCi, radiochemical purity 95%) were obtained from the microfluidic device. The crude material was then purified, and > 100 mCi of the final product was obtained in an overall uncorrected radiochemical yield of 5 ± 1% (n = 3), relative to starting [¹⁸F]fluoride (end of bombardment), with high radiochemical purity (≥ 99%) and high specific activities (6 Ci/μmol) in 100 minutes. A clinical research study was carried out with [¹⁸F]T807, representing the first reported human imaging study with a radiopharmaceutical prepared by this technology.

MICROFLUIDIC CHEMISTRY is a rapidly growing field in the application of radiopharmaceuticals. This technology offers an alternative to traditional vessel-based methods and can provide an efficient and flexible platform for reaction optimization starting from relatively small amounts of radioactivity. The first on-chip generation of radiotracers, ¹ including a study with [¹⁸F]fluorodeoxyglucose suitable for preclinical positron emission tomography (PET) studies, ² inspired reports of over 50 labeled compounds synthesized using various microfluidic devices across a variety of radionuclides, including carbon 11, nitrogen 13, ³ fluorine 18, copper 64, gallium 68, and technetium 99m.^4,5 Microfluidics systems can be divided into two major categories: microvessel systems (MVSs), which are miniature versions of batch reactor modules, and microchannel systems (MCSs), which are reaction systems based on flow chemistry in a microenvironment. ⁴ A prototypical MVS device was recently used to prepare [¹⁸F]fallypride for human use. ⁶ Despite extensive preclinical imaging with radiotracers prepared by MCSs, there is still no reported human PET imaging study enabled by this technology. We and others recently demonstrated several hardware/software modifications and improvements to MCS, including a microfluidic flow hydrogenation platform, ⁷ a dose-on-demand ¹⁸F- tracer production,^8–10 a modification of integrated high-performance liquid chromatography (HPLC) purification and solid phase extraction (SPE) formulation, ¹¹ and validation of fluorine 18-labeled 3-fluoro-5-[(pyridin-3- yl)ethynyl] benzonitrile ([¹⁸F]FPEB), ¹² that may accelerate the widespread use of this technology for the development of PET radiopharmaceuticals. Fluorine 18-labeled 7-(6- fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([¹⁸F]T807) is a PET radiotracer for imaging paired helical filaments of tau.^13,14 We recently reported a simplified one-step radiosynthesis of [¹⁸F]T807 ¹⁵ that we are presently using for clinical research studies in subjects with dementias and traumatic brain injuries. The goal of this proof-of-concept work is to validate the production of [¹⁸F]T807 by microfluidic flow chemistry for use in a human imaging study.

Materials and Methods

Optimization and Production of [¹⁸F]T807 by Microfluidic Flow Chemistry

An integrated Advion NanoTek microfluidic system and a GE Tracerlab FX_FN (General Electric, Germany), as described by us, ¹² were used to perform the radiosynthesis of [¹⁸F]T807. Briefly, [¹⁸F]fluoride was trapped on an anion exchange resin (MP1, ORTG, Inc., Oakdale, TN), eluted with nBu₄NHCO₃ (0.075M aqueous solution, ABX GmbH, Radeberg, Germany), and azeotropically dried using a standard NanoTek drying macrosequence. Our modified N-tert-butoxycarbonyl (t-Boc) protected precursor, tert-butyl 7-(6-nitropyridm-3-yl)-5H- pyrido[4,3-b]indole-5-carboxylate ¹⁵ (1 mg in 400 μL dimethyl sulfoxide [DMSO], concentration 2.5 mg/mL), and dried [¹⁸F]nBu₄NF (resolubilized in 400 μL DMSO) were loaded into the storage loops (≈ 400 μL) and dispensed into the microfluidic flow reactor (4 m × 100 μm) at various temperatures (150–210°C) and total flow rates (40–60 μL/min) to optimize the reaction chemistry. The optimized chemistry was then converted to an automated macro for the batch production, and the ensuing reaction mixture was transferred into a dilution vial (precharged with 25 mL H₂O) and mixed under a stream of nitrogen. The diluted crude mixture was prepurified on a solid-phase extraction cartridge (Waters Oasis HLB Light) to remove DMSO and was subsequently transferred to a GE TracerLab FX_FN for HPLC purification and formulation. Reaction conditions, including reaction temperature and flow rate, as shown in Figure 1, were optimized using the NanoTek discovery mode according to the method of Chun and colleagues, ¹⁶ and radiochemical conversions were measured by radio-thin-layer chromatography (radioTLC) (90% MeCN/H₂O). The purification, formulation, and quality control of the radiopharmaceutical were carried out by our procedure. ¹⁵ Briefly, [¹⁸F]T807 was purified by a semipreparative column (X-Select HSS T3, 250 × 10.00 mm, 5 m) and eluted with 18% EtOH/H₂O by volume (pH 2, adjusted with HCl) at a flow rate of 5 mL/min. The product was collected at 22 minutes and reformulated into 10% EtOH in saline (10 mL) by an Oasis HLB Light SPE cartridge. The synthesis and quality control of the [¹⁸F]T807 are summarized in Table 1.

Figure 1.

Synthesis and reaction optimization of [¹⁸F]T807 synthesized by microfluidic flow chemistry. HPLC = high-performance liquid chromatography.

Table 1.

Summary and Quality Control of [¹⁸F]T807 Synthesis

Summary	Results (n = 3)
Synthesis time	≈ 55 min (NanoTek) and < 100 min ready for injection
Synthesis activity	435 ± 120 mCi (16.1 ± 4.4 GBq; > 95% radiochemical purity and transferred to GE Tracerlab FXFN)
Synthesis yield (crude)	17 ± 1% from NanoTek, uncorrected, relative to starting [18F]fluoride (EOB)
Purification and formulation	≈ 45 min
Product activity	118 ± 19 mCi (4.4 ± 0.2 GBq; ready for injection)
Radiochemical yield	5 ± 1%, uncorrected, relative to starting [18F]fluoride (EOB)
Quality Control	Results (n = 3)	Specifications
Radiochemical identity	[18F]T807 < ± 10% of T807 reference standard	[18F]T807 ≤ ± 10% of T807 reference standard
Radiochemical purity	≥ 99%	≥ 95%
Chemical purity	5.14 μg ± 1.06 μg of T807	Not greater than 13 μg of T807
	0.49 μg ± 0.29 μg of total impurities	Not greater than 3 μg of total impurities
Specific activity	6.0 ± 1.4 Ci/μmol	≥ 0.4 Ci/μmol
Residual solvent analysis	DMSO 0.44 ± 0.16 mg/mL	DMSO ≤ 5 mg/mL
	Acetone ND	Acetone ≤ 5 mg/mL
	Acetonitrile ND	Acetonitrile ≤ 0.4 mg/mL
	Ethanol 9.2% v/v ± 2.5%	Ethanol 10% v/v ± 10%
pH assay	5–5.5	4.5–8
Radionuclidic Identity: photopeak	> 99.5% emission@511 KeV, 1.022 MeV, or Compton scatter peaks	≥ 99.5% emission@511 KeV, 1.022 MeV, or Compton scatter peaks
Radionuclidic Identity: half-life	108.1 ± 1.5 min	105-115 min
Sterile filter integrity test	51.6 ± 1.5 psi	≥ manufacturer's specification of 46 psi
Endotoxin analysis	< 5 EU/mL	Not greater than 17.5 EU/mL
Sterility testing	No evidence of growth at 14 days posti	noculation No evidence of growth at 14 days postinoculation

DMSO = dimethyl sulfoxide; EOB = end of bombardment; ND = not detected.

Image Acquisition Protocol and Data Analysis

A 32-year-old male subject was recruited from the community for participation in this research study. The protocol was approved by the Institutional Review Board at Partners Healthcare and the Radioactive Drug Research Committee at Massachusetts General Hospital. The subject provided written informed consent after the purpose, nature, and potential complication of the studies were explained and before participating in the experiments.

The subject underwent dynamic PET imaging on the ECAT EXACT HR+ scanner. After a transmission scan with rotating ⁶⁸Ge line sources, collection of emission data began concurrent with slow intravenous bolus administration of 5.13 mCi (190 MBq) of [¹⁸F]T807 prepared by the microfluidic synthesis procedure described herein. PET data were reconstructed using filtered back-projection and then normalized by injected dose and subject body weight to produce images in units of standardized uptake value (SUV). A Ti-weighted MPRAGE structural image of the brain was acquired on the Siemens Skyra 3 T magnetic resonance scanner. A PET image consisting of data integrated over the 0 to 60-minute time period after tracer injection was rigidly aligned to the subject's MRI, which was in turn registered to a standardized space.

Results

The microfluidics platform was used to conduct rapid screening of reaction parameters (Figure 1, entries 1–5) by dispensing a small amount of precursor and dried [¹⁸F] nBu₄NF simultaneously in a flow reactor. In general, fluorine 18 incorporation (by radioTLC) increased from the initial < 5% at 150°C to the highest 74% at 210°C using tetrabutylammonium bicarbonate (TBAB) and precursor (2.5 mg/mL) using a total flow rate of 40 mL/min (1:1 ratio), which represents the optimal conditions (entry 5). Another commonly used base system, K₂₂₂/K₂CO₃, was also tested and proved to be inferior and is likely attributed to decreased stability over high temperatures (> 190°C). Based on this result, three consecutive productions of [¹⁸F]T807 were carried out to validate this radiopharmaceutical for human use. Uncorrected radiochemical yields of 17 ± 1% of isolated crude [¹⁸F]T807 (< 500 mCi, 18.5 GBq, radiochemical purity 95%) were obtained from the microfluidic device. The crude material was then purified using the GE Tracerlab FX_fn, and > 100 mCi (3.7 GBq) of the final formulated material was obtained in overall uncorrected radiochemical yields of 5 ± 1% (n = 3), relative to starting [¹⁸F]fluoride at the end of bombardment (EOB), with high specific activities (6.0 ± 1.4 Ci/mmol; 222 ± 52 GBq/mmol) within 100 minutes.

Formulated [¹⁸F]T807 maintained stability, as measured by radioHPLC and radioTLC, as well as clarity and a pH of 5.5 over a period of 6 hours. The half-life was verified to be 109.7 minutes by a dose calibrator. No long-lived isotopes were observed (5 days), as determined by analysis on an high-purity germanium (HPGe) detector after ¹⁸F decay. The integrity of the final filter was demonstrated by a bubble-point filter test (> 46 psi). The formulated product was sterile and nonpyrogenic. Volatile organic compound analysis was carried out by gas chromatography with flame ionization detector showing residual acetone, CH₃CN, and DMSO below the lower limit of detection, thereby exceeding International Conference on Harmonisation requirements. The synthesis and quality control of [¹⁸F]T807 synthesis are summarized in Table 1. Using this microfluidic device, [¹⁸F]T807 is successfully validated for human PET studies and meets all Food and Drug Administration and U.S. Pharmacopeial Convention (USP) requirements for a PET radiopharmaceutical.

Intravenous administration of [¹⁸F]T807 had no adverse effects, as observed by study staff or reported by the subject. The PET image showed good signal in brain with relatively high uptake in gray matter relative to white matter (Figure 2). The characteristics of the image data acquired with the tracer prepared by microfluidic methods were consistent with those that we observed in other subjects using [¹⁸F]T807 as prepared by our alternative synthesis. ¹⁵

Figure 2.

Axial, coronal, and sagittal slices from the human PET image acquired over the 0 to 60-minute interval after injection of [¹⁸F]T807 prepared by microfluidic flow chemistry. PET images are overlaid on the subject's T₁-weighted structural magnetic resonance image. SUV = standardized uptake value.

Discussion

The original synthesis of [¹⁸F]T807 involves a semiautomated two-step reaction^13,14 and uses 7-(6-nitropyridin-3- yl)-5H-pyrido[4,3-b]indole as the precursor. We recently developed a one-step radiosynthesis [¹⁸F]T807 using a new N-tert-butoxycarbonyl (t-Boc) protected precursor, with an isocratic HPLC purification using a GE TRACERlab FXFN module for routine human use. ¹⁵ Herein we report the synthesis of [¹⁸F]T807 produced by a microfluidic device as a proof of concept that this technology is suitable for human PET imaging studies.

Fluorine 18-labeled T807 prepared by this flow chemistry method did not show signs of radiolysis during the radiofluorination or after the reformulation, in spite of the high radioactivity concentration (> 3 Ci, 111 GBq of starting fluorine 18 in 400 μL in a storage loop). The radiochemical yields herein (5 ± 1%, uncorrected for decay) were lower than that of our automated method (14 ± 3%, uncorrected). ¹⁵ Although radioactivity losses could be minimized by further optimization of the purification processes, given that more than sufficient quantities (> 100 mCi) of [¹⁸F]T807 were prepared suitable for injection, no further development was carried out for this proof-of-concept study. Routine use of this technology for the production of PET radiopharmaceuticals in a regulated environment would require the integration of a purification and formulation module to the microfluidic system.

Following administration of [¹⁸F]T807 into a human subject, high radiotracer uptake was seen in the brain (SUV > 2 in many regions; see Figure 2). We have already imaged over 100 subjects with this radiotracer at our laboratories with our recently reported methodology ¹⁵ for Alzheimer disease and other tauopathies (to be published elsewhere), and the brain uptake in the present work is consistent with our experience with [¹⁸F]T807 uptake. This work demonstrates that microfluidic flow chemistry systems are viable platforms for synthesizing PET radiopharmaceuticals suitable for human PET imaging studies.