@misc{oai:repo.qst.go.jp:00072869,
 author = {Kikuchi, Tatsuya and Okada, Maki and Ishii, Hideki and Nengaki, Nobuki and Ono, Maiko and Okamura, Toshimitsu and Minamimoto, Takafumi and Higuchi, Makoto and Zhang, Ming-Rong and 菊池 達矢 and 岡田 真希 and 石井 英樹 and 念垣 信樹 and 小野 麻衣子 and 岡村 敏充 and 南本 敬史 and 樋口 真人 and 張 明栄},
 month = {Jul},
 note = {Introduction
[11C]PBB3, a Tau imaging agent, is rapidly metabolized to form a sulfate conjugate ([11C]PBB3-sulfate) in peripheral tissues by sulfotransferase (SULT).1 After the administration of [11C]PBB3 to mice, [11C]PBB3-sulfate was observed also in the brain. The previous report concluded that it was due to the influx of [11C]PBB3-sulfate from blood to brain. However, it is known that SULT is expressed in the brain and [11C]PIB which possesses 6-hydroxybenzothiazole as a fundamental structure (Fig 1) is sulfated in the rat brain.2 It was therefore expected that [11C]PBB3, a [11C]PIB derivative, would be sulfated in the brain. In this study, the sulfate conjugation of [11C]PBB3 in the brain was investigated because the conjugation might affect the kinetic analysis of Tau imaging with [11C]PBB3. In addition, the hydrolysis of [11C]PBB3-sulfate to form parent [11C]PBB3 in the brain was investigated as sulfate conjugates can be hydrolyzed by sulfatase which is expressed in the brain.

Materials & Methods
Sulfate conjugation of [11C]PBB3 in the brain was evaluated by adding [11C]PBB3 and a sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the brain homogenate sample. In mice samples (cerebrum, C57BL/6, 8 wo, n=3), reactions were carried out at 37°C for 30 min. In samples of a rhesus monkey (frontal cortex, Macacca mulatta, male, 23 yo), reactions were carried out at 37°C and the conjugates generated were analyzed at 5, 15, 30 min after starting reaction. In addition to the crude homogenate (around 0.2 g/mL for mice, 0.16 g/mL for monkey), the sulfate conjugation in the supernatant of homogenate was investigated. The conjugation was analyzed using radio-TLC. Similarly, [11C]PBB3-sulfate, which was prepared by O-sulfation of [11C]PBB3 using sulfur trioxide pyridine complex, was incubated in the mice brain homogenate at 37°C for 30 min to evaluate its hydrolysis.

Results
The sulfate conjugation of [11C]PBB3 was observed in the mice brain samples with 30 min reaction time, and only 7.7±0.44% and 9.2±0.30% of [11C]PBB3 was conjugated in the crude homogenate and supernatant, respectively (Fig 2A). Meanwhile, [11C]PBB3-sulfate was slowly hydrolyzed to form [11C]PBB3 in the brain sample (3.1±0.13% in the crude homogenate and 3.7±0.20% in the supernatant, Fig 2B).
The conjugation rates of [11C]PBB3 in the monkey brain samples were much faster than that in the mice brain samples. The conjugation in the crude homogenate of monkey brain followed first order manner (r2=1.0) up to 91% decrease of [11C]PBB3 and the rate was estimated to be 0.49 min–1(g/mL)–1. The first order kinetics suggested that the hydrolysis rate of [11C]PBB3-sulfate in the monkey brain was much slower than the conjugation rate. Indeed, the conjugation rate and the hydrolysis rate in the supernatant prepared from 0.16 g/mL of monkey brain homogenate were estimated to be 0.35 min-1 and 0.013 min-1, respectively (Fig 3).

Discussion
In mice, [11C]PBB3 slowly but significantly underwent sulfate conjugation in the brain. Therefore, a part of [11C]PBB3-sulfate observed in the mouse brain after [11C]PBB3 administration would be the metabolite generated in the brain. On the other hand, the sulfate conjugation of [11C]PBB3 was quite rapid and apparently irreversible in the monkey brain samples. 
In this study, large Interspecies differences regarding the sulfate conjugation of [11C]PBB3 in the brain was observed. Therefore, further investigation about the conjugation of [11C]PBB3 in the human brain would be required.

References
1.Hashimoto H, Kawamura K, Takei M et al. [2015] Nucl. Med. Biol. 42: 905–910
2.Cole GB, Keum G, Liu J et al. [2010] Proc. Natl. Acad. Sci. USA 107: 6222–6227, The XII International Symposium of Functional Neuroreceptor Mapping of the Living Brain},
 title = {Sulfate conjugation of [11C]PBB3, a Tau imaging agent, in the brain},
 year = {2018}
}