Automated Production and Quality Testing Established for Two Alpha-Synuclein PET Tracers

A multidisciplinary research team has developed standardized, automated radiosynthesis protocols and rigorous quality control testing procedures for two fluorine-18 labeled radiopharmaceuticals designed to image pathological alpha-synuclein aggregates in clinical investigations. The two positron emission tomography imaging probes, designated as [18F]SPAL-T-06 and [18F]C05-05, were successfully manufactured using an automated synthesizer system, achieving high radiochemical purity, sufficient molar activity, and reproducible yields suitable for routine clinical application. By validating the radiosynthetic parameters and formulation processes, the researchers established a reliable technical foundation intended to facilitate multicenter technology transfer and support standardized diagnostic imaging of neurodegenerative alpha-synucleinopathies.

Both radiopharmaceuticals were engineered to selectively visualize intracellular alpha-synuclein inclusions, which represent pathological hallmarks in conditions such as multiple system atrophy and Parkinson’s disease. Previous diagnostic work by the research group established the utility of carbon-11 labeled [11C]PBB3 and fluorine-18 labeled [18F]PM-PBB3, also known as [18F]APN-1607 or [18F]florzolotau, for imaging tau aggregates in human tauopathies. Building upon those tracer platforms, clinical investigations recently demonstrated that PET scans using [18F]SPAL-T-06 deliver high-contrast visualization of alpha-synuclein deposits in patients with multiple system atrophy, enabling individual-level diagnostic interpretation, while [18F]C05-05 has proven effective for clinical assessments in Parkinson’s disease and related disorders alongside evaluated human radiation dosimetry.

The molecular design of these second-generation probes relies on a specific structural modification of the conjugated backbone that dramatically shifts binding selectivity from tau and amyloid-beta fibrils toward alpha-synuclein. Replacing the (2E,4E)-hexa-2,4-dien linker found in the PBB3 series with an (E)-hex-2-en-4-yne linker generated C05-05 and SPAL-T-06, producing an altered backbone twist angle at the minimum energy conformation that matches the distinct geometry of alpha-synuclein fibril binding pockets. Pharmacological assays confirmed this structural optimization, showing homologous binding blockade half-maximal inhibitory concentrations of 1.5 nanomolar for C05-05 and 1.6 nanomolar for SPAL-T-06, which represent approximately forty-fold and eighteen-fold lower values than those observed for PBB3 and PM-PBB3.

Automated production of both tracers was implemented using an automated multipurpose radiosynthesis platform coupled to an institutional fluorine-18 labeling synthesizer. Fluorine-18 was generated as aqueous fluoride via the 18O(p,n)18F nuclear reaction in a cyclotron employing eighteen-megaelectronvolt proton bombardment of enriched target water containing greater than 98 atomic percent oxygen-18. The cyclotron-produced fluoride was extracted and isolated from target water through solid-phase extraction using an anion-exchange cartridge, which had been sequentially preconditioned with ethanol, water for injection, potassium carbonate solution, and water for injection prior to radiochemical trapping.

The trapped fluoride was subsequently eluted into the reaction vessel using a solution containing potassium carbonate and the phase-transfer catalyst Kryptofix 222 dissolved in aqueous acetonitrile. Complete solvent removal and drying of the fluoride complex were accomplished by gradual thermal evaporation at temperatures ranging from 80 to 130 degrees Celsius over a 15 to 30 minute drying sequence. Direct nucleophilic fluorination was then initiated by adding two milligrams of the appropriate tetrahydropyranyl-protected tosylated precursor dissolved in 0.3 milliliters of anhydrous dimethyl sulfoxide, followed by heating at 130 degrees Celsius for 15 minutes.

Deprotection parameters were systematically optimized after initial trials using hydrochloric acid under reaction conditions identical to [18F]PM-PBB3 synthesis yielded an unexpectedly low non-decay-corrected radiochemical yield of only 4.7 percent for [18F]SPAL-T-06 at the end of synthesis. Substituting hydrochloric acid with 50 percent aqueous trifluoroacetic acid increased the non-decay-corrected yield to 7.9 percent. Lowering the cleavage temperature from 90 to 70 degrees Celsius to prevent excessive vaporization below the 72-degree boiling point of trifluoroacetic acid increased the yield to 9.9 percent, and further raising the trifluoroacetic acid concentration to 70 percent achieved an 11.4 percent yield before quenching with a sodium acetate and dimethyl sulfoxide mixture.

Under these refined deprotection conditions, routine automated runs of [18F]SPAL-T-06 achieved an average non-decay-corrected radiochemical yield of 11 plus or minus 4.6 percent across 19 production batches, with molar activities of 390 plus or minus 230 gigabecquerels per micromole and synthesis durations spanning 76 to 89 minutes from the end of cyclotron irradiation. For [18F]C05-05, produced under the same reaction parameters, the non-decay-corrected radiochemical yield reached 20 plus or minus 6.8 percent across 37 batches, exhibiting molar activities of 280 plus or minus 130 gigabecquerels per micromole and synthesis durations between 89 and 99 minutes, with both compounds exceeding 99 percent radiochemical purity.

The authors noted that the observed radiochemical yields of [18F]SPAL-T-06 and [18F]C05-05 were lower than the 25 percent yield typically obtained for [18F]PM-PBB3 due to fundamental chemical reactivity differences. Unlike [18F]PM-PBB3, which incorporates two carbon-carbon double bonds, both [18F]SPAL-T-06 and [18F]C05-05 possess a conjugated carbon-carbon triple bond that is more electron-deficient and more susceptible to hydration under acidic deprotection conditions. Furthermore, the lower yield of [18F]SPAL-T-06 relative to [18F]C05-05 was attributed to electronic differences between the pyrazine ring of the former and the pyridine ring of the latter.

Purification of the crude reaction mixtures was conducted using semi-preparative reverse-phase high-performance liquid chromatography on an Inertsil ODS-HL column with ultraviolet absorbance monitoring at 390 nanometers, yielding retention times of approximately 15.6 minutes for [18F]SPAL-T-06 and 16.1 minutes for [18F]C05-05. The purified fractions were collected into flasks preloaded with polysorbate 80 and ascorbic acid to prevent radiolytic degradation, evaporated under reduced pressure at 300 degrees Celsius, reconstituted in physiological saline, and sterilely filtered through a Millex-GV membrane inside a class-A isolator, where automated bubble point pressure tests exceeding 291 kilopascals confirmed filter membrane integrity.

Comprehensive quality control testing of three independent validation batches confirmed that both radiopharmaceutical preparations complied with all pre-established specifications under Japanese Pharmacopoeia standards and Japanese Society of Nuclear Medicine Good Manufacturing Practice guidelines. Analytical high-performance liquid chromatography confirmed identity against non-radioactive reference standards, with retention times of 11.9 minutes for [18F]SPAL-T-06 and 10.3 minutes for [18F]C05-05. Gas chromatography verified that residual solvents remained within safe limits, thin-layer chromatography confirmed Kryptofix 222 removal below 40 micrograms per milliliter, gamma spectroscopy verified radionuclidic purity, and endotoxin assays satisfied safety thresholds.

Photostability investigations demonstrated that both radioligands are vulnerable to rapid photoisomerization when exposed to conventional fluorescent lighting within a standard laboratory setting. Exposure to standard fluorescent light between 760 and 930 lux caused the radiochemical purity of each tracer to drop to approximately 86 percent within 10 minutes and decline further to approximately 64 percent after 60 minutes. Although this degradation represents greater stability than that of [18F]PM-PBB3, which falls to 24 percent purity within 5 minutes because of its diene structure, fluorescent light exposure still results in unacceptable isomerization for clinical preparations.

In contrast, shielding the radiopharmaceuticals under ultraviolet-cutoff yellow lighting with wavelengths above 500 nanometers completely prevented photoisomerization, maintaining radiochemical purity above 99 percent after 60 minutes of illumination. Samples maintained in light-shielded containers without direct illumination also retained radiochemical purities above 99 percent for at least 180 minutes following formulation. Consequently, the researchers concluded that all post-deprotection synthesis operations, formulation steps, and quality control procedures must be strictly executed under ultraviolet-cutoff lighting in natural light-shielded environments to guarantee the stability and diagnostic reliability of both radiotracers.

Subject of Research: Automated radiosynthesis and quality control of alpha-synuclein PET radiopharmaceuticals

Article Title: Radiosynthesis and quality control testing of two 18F-labeled radiopharmaceuticals for α-synuclein imaging in clinical investigations

Article References: Hashimoto, H., Ohkubo, T., Kawamura, K., Fujishiro, T., Arashi, D., Sato, T., Seki, T., Muto, M., Tsukagoe, K., Ogawa, M., Kurihara, Y., Nengaki, N., Mori, W., Fujinaga, M., Higuchi, M., & Zhang, M.-R. (2026). Radiosynthesis and quality control testing of two 18F-labeled radiopharmaceuticals for α-synuclein imaging in clinical investigations. EJNMMI Radiopharmacy and Chemistry, 11(1), Article 76. https://doi.org/10.1186/s41181-026-00480-w

Image Credits: AI Generated

DOI: 10.1186/s41181-026-00480-w

Keywords: PET imaging, alpha-synuclein, radiopharmaceuticals, radiosynthesis, quality control, fluorine-18, photoisomerization, neuroimaging, quality, control, testing, F-labeled