An one-pot two-step automated synthesis of [18F]T807 injection, its biodistribution in mice and monkeys, and a preliminary study in humans

Article abstract of DOI:10.1371/journal.pone.0217384

[¹⁸F]T807 is a potent tau protein imaging agent. In order to fulfill the demand from preclinical and clinical studies, we developed an automated one-pot two-step synthesis of this potent tau imaging agent and studied its stability, and dosimetr

Full text of DOI:10.1371/journal.pone.0217384

RESEARCH ARTICLE
An one-pot two-step automated synthesis of
[18F]T807 injection, its biodistribution in mice
and monkeys, and a preliminary study in
humans
Ya-Yao Huang 1*, Ming-Jang Chiu2,3,4,5,6, Ruoh-Fang Yen1,2,7, Chia-Ling Tsai1, Hao-
ID
Yu Hsieh8, Ching-Hung Chiu1, Chi-Han Wu2, Ling-Wei Hsin2,8, Kai-Yuan Tzen1,2, Cheng-
1,2,9
Yi Cheng9, Kuo-Hsing Ma10, Chyng-Yann Shiue
*
ID
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
1 PET Center, Department of Nuclear Medicine, National Taiwan University Hospital, Taipei, Taiwan,
2 Molecular Imaging Center, National Taiwan University, Taipei, Taiwan, 3 Departments of Neurology,
National Taiwan University Hospital, Taipei, Taiwan, 4 Institute of Brain and Mind Sciences, College of
Medicine, National Taiwan University, Zhongzheng Dist., Taipei, Taiwan, 5 Department of Psychology,
National Taiwan University, Taipei, Taiwan, 6 Graduate Institute of Biomedical Engineering and Bio-
informatics, National Taiwan University, Taipei, Taiwan, 7 Department of Radiology, National Taiwan
University College of Medicine, Taipei, Taiwan, 8 School of Pharmacy, College of Medicine, National Taiwan
University, Zhongzheng Dist., Taipei, Taiwan, 9 PET Center, Department of Nuclear Medicine, Tri-Service
General Hospital, Neihu, Taipei, Taiwan, 10 Department of Biology and Anatomy, National Defense Medical
Center, Taipei, Taiwan
OPEN ACCESS
* careyyh@ntuh.gov.tw (Y-YH); shiue@ntuh.gov.tw (C-YS)
Citation: Huang Y-Y, Chiu M-J, Yen R-F, Tsai C-L,
Hsieh H-Y, Chiu C-H, et al. (2019) An one-pot two-
step automated synthesis of [18F]T807 injection, its
biodistribution in mice and monkeys, and a
preliminary study in humans. PLoS ONE 14(7):
e0217384. https://doi.org/10.1371/journal.
pone.0217384
Abstract
[18F]T807 is a potent tau protein imaging agent. In order to fulfill the demand from preclinical
and clinical studies, we developed an automated one-pot two-step synthesis of this potent
tau imaging agent and studied its stability, and dosimetry in mice and monkeys. We also
conducted a preliminary study of this imaging agent in humans. Using this one-pot two-step
method, the radiochemical yield (RCY) of [18F]T807 was 20.5 ± 6.1% (n = 15) at the end of
bombardment (EOB) in a synthesis time of 70±5 min. The chemical and radiochemical puri-
ties were >90% and the specific activities were 151 ± 52 GBq/μmol. The quality of [18F]T807
synthesized by this method met the U.S. Pharmacopoeia (USP) criteria. The stability test
showed that the [18F]T807 injection was stable at room temperature for up to 4 h after the
end of synthesis (EOS). The estimated effective dose of the [18F]T807 injection extrapolated
from monkeys was 19 μSv/MBq (n = 2), while the estimated effective doses of the [18F]T807
injection extrapolated from fasted and non-fasted mice were 123 ± 27 (n = 3) and 94 ± 19
(n = 4) μSv/MBq, respectively. This one-pot two-step automated method produced the [18F]
T807 injection with high reproducibility and high quality. PET imaging and radiation dosime-
try evaluation in mice and Formosan rock monkeys suggested that the [18F]T807 injection
synthesized by this method is suitable for use in human PET imaging studies. Thus, this
method could fulfill the demand for the [18F]T807 injection in both preclinical and clinical
studies of tauopathies, especially for nearby study sites without cyclotrons.
Editor: Chin-Tu Chen, University of Chicago,
UNITED STATES
Received: April 3, 2018
Accepted: May 11, 2019
Published: July 1, 2019
Copyright: © 2019 Huang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by National
Taiwan University Hospital (NTUH 108-A133 and
NTUH 108-S4177) and Ministry of Science and
Technology (MOST 108-2321-B-002-002 – and
MOST 107-2314-B-002- MY2).
Competing interests: The authors have declared
that no competing interests exist.
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Introduction
Alzheimer’s disease (AD) is one of the most frequent causes of death and disability worldwide,
and has a significant clinical and socio-economic impact. It is the most common form of
dementia in individuals over 65 y and accounts for 50–60% of dementia cases. AD is character-
ized by the degeneration of neurons in the hippocampus and cortex, and the appearance of β-
amyloid plaques and neurofibrillary tangles, both of which appear many years before the onset
of symptoms of cognitive impairment [1–5]. Although the precise cause of AD remains
unclear, it is most likely due to multiple etiologies such as neuronal apoptosis, inflammatory
responses, and alterations in various receptors and enzymes. Thus, several PET imaging agents
that target multiple mechanisms such as various receptors, enzymes, and β–amyloids have
been developed [6, 7] for monitoring the response of AD drug therapy non-invasively and
facilitating AD drugs development. Among these, β–amyloid imaging agents have improved
the ascertainment of early stages of AD. Unfortunately, the levels of β–amyloid do not corre-
late as closely to the clinical phenotype as do tau proteins [8–10]. Thus, for the past few years,
development of tau imaging agents is among the most active areas of research in molecular
imaging of neurodegenerative diseases. This has led to the development of [18F]T807 ([18F]
AV-1451) [11, 12], [18F]T808 ([18F]AV-680) [13, 14], [11C]PBB3 [15], [18F]THK5105 [16, 17],
[18F]THK523 [18–20], [18F]THK5117 [16, 21], [18F]THK5351 ([18F]GE-216) [22], [18F]
RO6958548, [11C]RO6931643, [11C]RO6924963 [23–27] and most recently, [18F]MK-6240
[28] and [18F]PI-2620 [29] as tau imaging agents for clinical studies of AD pathophysiology.
Among these, [18F]T807 is the most widely used tau imaging agent in clinical studies [12, 30–
34]. In addition to several AD clinical studies, [18F]T807 has also been applied to studies of
chronic traumatic encephalopathy [35], frontotemporal dementia (FTD) [35], Parkinson’s dis-
ease [36], and primary progressive aphasia [37].
[18F]T807(1) was first synthesized by a one-step method using a modified Siemens Explora
radiosynthesis module. Fluorination of its nitro-precursor (T807P, 2) with [18F]Fluoride ([18F]
F-) followed by reduction with Fe powder and purification with HPLC gave [18F]T807 in 29–
47% yield (EOB) (Fig 1) in a synthesis time of 90–93 min from EOB [11, 12]. However, when
[18F]T807 was synthesized with a home-built automated synthesis module, its RCY was only
´
5–10% (EOB) in a synthesis time of 60–70 min from EOB [38]. Meanwhile, Bramoulle et al.
[39] have modified the semi-preparative HPLC purification conditions without using Fe pow-
der as a reducing agent and successfully produced [18F]T807 with TRACERlab FXFN Pro syn-
thesizer in 14% yield (EOB) in a synthesis time of 75 min from EOB. However, there was no
mention of either the chemical or radiochemical purity of the [18F]T807 product synthesized
by this method. Recently, Shoup et al. [40] have reported an improved one-step synthesis of
[18F]T807 using a t-Boc-protected nitro-precursor (t-Boc-T807P or AV-1628, 3) and on-line
acidic de-protection of t-Boc-protecting group, resulting in a RCY of 16–25% (EOB) in a syn-
thesis time of 60 min from EOB (Fig 2) (Method A). However, we have found that this on-line
acidic de-protection of the t-Boc-protecting group was somewhat difficult to reproduce.
Fig 1. Original one-step radiosynthesis of [18F]T807(1).
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 2. Improved two-step radiosynthesis of [18F]T807(1).
https://doi.org/10.1371/journal.pone.0217384.g002
In order to facilitate its widespread use for tauopathies and traumatic brain injuries [41,
42], it is imperative to have a reliable, fully automated high yield and GMP-compliant
manufacturing process method available for the production of the [18F]T807 injection (1).
Thus, we have adopted Shoup’s method [40] with minor modifications to fully automate the
synthesis of the [18F]T807 injection under GMP-compliance using a TRACERlab FXFN mod-
ule (GE Healthcare, Milwaukee, WI) in high quality and high reproducibility, and study its
whole-body biodistribution and dosimetry estimations in ICR mice, Formosan rock monkeys
and in humans. We herein report a GMP-compliant high reliability fully automated synthesis
of [18F]T807 using a TRACERlab FXFN module along with its whole-body biodistribution in
ICR mice and Formosan rock monkeys, and the validation of this radiotracer in human. Part
of this work has been present in abstract form [43].
Material and methods
Chemicals and reagents
All chemicals and reagents were obtained from commercial vendors and were used as received
without further purification. All Sep-Pak cartridges were obtained from Waters (Milford, MA,
USA) and highly enriched (> 98.0 Atom%) [18O]H2O was purchased from Huayi Isotopes Co.
(Changshu, China). Aqueous [18F]F− was produced in our PET Trace cyclotron (GE Medical
Systems, Uppsala, Sweden) via an 18O(p, n)18F nuclear reaction. The precursor (tert-butyl-7-
(6-nitropyridin-3-yl)-5H-pyrido[4,3-b]indole-5-carboxylate, 3) and the non-radioactive
authentic standard T807 (1a) were synthesized by literature method [44] with minor modifica-
tions as shown in Fig 3. The identity of 3 was confirmed by 1H-NMR (S1 Fig), 13C-NMR (S2
Fig 3. Synthesis of precursor (3) and the non-radioactive standard (1a) of [18F]T807 (1).
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig), and mass spectra (S3 Fig). The chemical purity of compounds 3 was determined by
reverse-phase high-performance liquid chromatography, and was higher than 95% (S4 Fig)
(please see Supporting Information).
Fully automated production of the [18F]T807 injection using a FXFN
module
Prior to delivery of [18F]fluoride to the TRACERLab FXFN synthesis module, each vial was
filled with appropriate solvent and reagent as shown in Fig 4.
Vial 1: K2CO3 (4.7 mg in 0.25 mL H2O) and K2.2.2. (14.7 mg in 0.25 mL ACN).
Vial 2: Acetonitrile (ACN) (1.0 mL).
Vial 3: Precursor (1mg) in 0.5 mL DMSO.
Vial 4: 0.4 mL of 1 N HCl(aq)
Vial 5: 0.5 mL of 2 M NaOAc(aq)
Vial 6: 0.35 mL of EtOH/21 mM sodium phosphate (8/92).
Product bottle: 0.13 mL of 7% NaHCO3(aq)
.
.
.
V14 was directly connected to a Sep-Pak Alumina N Plus Long cartridge and then con-
nected to the tubing passing through the fluid detector for semi-preparative HPLC
purification.
The automated synthesis module was operated in the following sequence:
1. At the end of bombardment, aqueous [18F]F− in 1.4 mL of [18O]H2O was transferred from
the target into a [18O]H2O collection vial with helium purge.
2. Opened V10; aqueous [18F]F− was passed through a Sep-Pak QMA Plus Light cartridge and
V11 under vacuum. [18O]H2O was recovered in the [18O]H2O recovery vial and the [18F]F−
was trapped on a Sep-Pak QMA Plus Light cartridge.
Fig 4. Modified TRACERLab FXFN module for [18F]T807 radiosynthesis.
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GMP-compliant [18F]T807 injection for preclinical and clinical use
3. Opened V1, V13, and V24. K2CO3/K2.2.2 solution in Vial 1 was passed through V1, V10,
QMA, V11, and V13. [18F]F− was collected in Reaction Vessel.
4. Closed V1, V13, and V14. Opened V24 and evaporated the solution in Reaction Vessel at
110˚C for 3 min under a stream of helium from V20.
5. Flushed ACN in Vial 2 into Reaction Vessel with helium. Closed V2, V13, and V14. Opened
V24, and performed azeotropic distillation of the remaining H2O with a stream of helium
from V20.
6. Flushed precursor solution in Vial 3 into Reaction Vessel with helium. Closed V2, V3, V13,
V14, V20, and V24. Heated the solution at 130˚C for 10 min.
7. Flushed HCl(aq) in Vial 4 into Reaction Vessel with helium. Closed V4, V13, V14, V20, and
V24. Heated the solution at 90˚C for 10 min.
8. Flushed NaOAc(aq) in Vial 5 and EtOH/sodium phosphate in Vial 6 into Reaction Vessel
with helium. Closed V5, V6, V13, V14, V20, and V24. Stirred the solution for 1 min at
50˚C.
9. Opened V14, passed the reaction mixture in the Reaction Vessel through a Sep-Pak Alu-
mina N cartridge with a stream of helium from V20 and immediately injected it into semi-
preparative HPLC (Phenomenex Luna(2) HILIC, 5 μm, 10 × 250 mm, 0.1% HCl(aq), 254
nm, 3 mL/min).
10. At approximately 18 min post-injection, the [18F]T807 peak was collected for 3 min and
the solution was transferred through V18 into the product bottle, which contained 7%
NaHCO3(aq)
.
11. Opened V16 and flushed the [18F]T807 solution in the final product bottle, which con-
tained 0.13 mL of 7% NaHCO3(aq) and 9 mL of 0.1% HCl(aq), through a 0.2 μm sterile filter
(Millex LG; Millipore, Billerica, MA, USA), into a sterile multi-injection vial with a stream
of helium from V22.
Quality control and stability tests of the [18F]T807 injection
The quality control (QC) tests of the [18F]T807 injection were performed in accordance with
the USP for radiopharmaceuticals, which include visual inspection, pH, half-life of radionu-
clide, radionuclidic purity, radiochemical purity, chemical purity, residual K2.2.2, residual sol-
vents, bacterial endotoxins, filter integrity, and sterility test as tabulated in Table 1.
The radiochemical and chemical purities of the [18F]T807 injection were analyzed with a
HPLC system (Agilent 1100 series) equipped with a Bioscan FC3300 flow count radioactivity
detector (2” × 2” pinhole) and an UV detector (254 nm) using a Phenomenex Luna HILIC col-
umn (5 μm, 4.6 × 250 mm) with 10% EtOH (pH = 2, adjusted with HCl) as mobile phase and a
flow rate of 1 mL/min. The radiochemical purity of [18F]T807 was further analyzed with radio
TLC (Silica gel 60 F254 plate, NH4OH/MeOH/CH2Cl2 (0.8/5/95)) and detected with a Raytest
miniGita radio-TLC scanner (Raytest, Straubenhardt, Germany).
The residual organic solvents in the [18F]T807 injection were analyzed by gas chromatogra-
phy (GC) equipped with a flame ionization detector (FID) and an Agilent Column (HP Fast
Residual Solvent chromatography column, 30 m × 0.53 mm × 1 μm). Nitrogen was used as a
carrier gas with a flow rate of 2 mL/min. The split/splitless injector was set at 20:1, the initial
oven temperature was kept at 40˚C for 1 min followed by an increase of temperature at 14˚C/
min to 110˚C and then at 30˚C/min to 125˚C. The oven temperature was kept at 125˚C for 2.5
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Table 1. Production and QC results of three consecutive productions of the [18F]T807 injection (n = 3).
Batch Production
Production Results
Run 1
77.4
5.3
Run 2
32.3
Run 3
92.9
6.8
Starting Activity (GBq)
Product Activity GBq)
1.7
Final Volume (ml)
9
9
9
Synthesis Time (min)
57
61
66
Specific Activity (GBq/μmol) (EOB)
542
323
408
QC Test
Acceptance Criteria
Clear, colorless solution
QC Results
Pass
7
Visual inspection
Pass
7
Pass
7
pH value
5–8
Residual K2.2.2 (μg/mL)
Radionuclidic Purity (%)
Half-life (min)
<50
<50
99.99
115
<50
99.99
114
<50
99.98
113
>99.5
110 5
Radiochemical Identity (min)
Radiochemical purity (%)
Residual solvent analysis
jRt-Rt(Reference)j�0.5
>90
0.25
0.10
0.23
93.12
ND
91.47
0.0116
ND
92.38
0.0069
0.047
ND
Acetone<0.5%
ACN<0.04%
DMSO<0.5%
EtOH<0.5%
<5
ND
0.0114
0.0069
0.12
0.0112
0.2469
0.26
<5
0.0113
0.26
Residual [18F]F- (%)
Bacterial endotoxins (EU/ml)
Filter integrity test (psi)
Sterility test
<17.5
<5
<5
>50
59
58
56
Sterile
Sterile
Sterile
Sterile
https://doi.org/10.1371/journal.pone.0217384.t001
min followed by an increase of temperature at 47.5˚C/min to 220˚C and kept at that tempera-
ture for 4 min. Hydrogen and air, at 35 and 250 mL/min, respectively, were used in the GC/
FID, with nitrogen (25 mL/min) as a make-up gas. The software Gina Star (Raytest, Strauben-
hardt, Germany) was used for data acquisition.
Measurement of bacterial endotoxins was carried out using a Limulus amoebocyte lysate
(LAL) test with Kinetic-QCL method (Lonza, Walkersville, MD, USA) and sterility test was
performed with the proven method meeting the requirement of USP.
The stability of the [18F]T807 injection at room temperature (18˚C~25˚C) was monitored
with both TLC and HPLC as described above for up to 4 h after EOS.
Whole-body biodistribution of the [18F]T807 injection in mice and
monkeys
Male ICR mice (20–30 g, 6–8 weeks old) and male Formosan rock monkeys (Macaca cyclopis)
(5 and 7.9 kg, n = 2) were used for this study. The animal study protocols of mice and monkeys
used for this study were approved by the Laboratory Animal Center of National Taiwan Uni-
versity (NTU) and National Defense Medical College (NDMC), respectively. Both animal cen-
ters have been granted full accreditation from the Association for Assessment and
Accreditation of Laboratory Animal Care International (AAALAC) since 2007. The animals
were housed and handled according to institutional guidelines.
ICR mice were obtained from NTU College of Medicine Laboratory Animal Center and
housed in the animal facilities at Department of Nuclear Medicine of National Taiwan Univer-
sity Hospital (Taipei, Taiwan) at a constant temperature of 26 2˚C, humidity of 30~70%, and
a controlled light/dark cycle (light from 7:00 AM to 7:00 PM). Mice were maintained on a
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GMP-compliant [18F]T807 injection for preclinical and clinical use
complete pellet diet and tap water for a period of 1 week prior to the studies. To compare the
effect of fasting on [18F]T807 whole body bio-distribution, the mice were either fasted for at
least 8 hours (n = 3) or non-fasted (n = 4) as the control counterparts.
The monkeys were bred at a temperature of 21~25 oC, humidity of 50~75%, ventilation of
8~15/hour, illumination from 7:00 AM to 7:00 PM, noise < 65 dB, and sanitation, to escalate
the homeostasis of animals in NDMC Animal Center (Taipei, Taiwan).
During scanning, mice were anesthetized with 2% isoflurane in oxygen and the monkeys
were immobilized with ketamine (2 mg/kg), anesthetized with 2% isoflurane via an endotra-
cheal tube and administered with atropine sulfate (2 mg IM) to minimize secretions during
the course of the experiment. Body temperature was kept constant at 37˚C with an additional
blanket. The heart rate, pO2 and pCO2 were checked every 10 min and kept in the normal
range throughout the imaging sessions. Whole-body transmission and emission scans of ICR
mice and Formosan rock monkeys were acquired with a small-animal Argus PET/CT scanner
(SEDECAL, Madrid, Spain) and a BIOGRAPH PET/CT scanner (Biograph Duo, Siemens,
USA), respectively. For mice studies, after the [18F]T807 injection (9 3 MBq), dynamic sino-
grams were produced for 90 min with 2 x 10 s, 2 x 20 s, 4 x 60 s, 2 x 240 s, 2 x 600 s, and 1 x 720
s frames. For the monkeys’ studies, after the [18F]T807 injections (107 MBq and 89 MBq), a
low dose CT scan (130 kVp, 50 mAs), a series of eight whole-body PET scans were performed
(15, 55, 95, 110, 155, 170, 215 and 230 min) post-injection. Total acquisition time was 240 min.
Each scan covered the monkey’s body from the head to the thigh and consisted of five to six
bed positions depending on the size of the monkey. In each bed position, data was acquired
for 2 min in 3D mode. The data was then reconstructed by OSEM (Ordered-Subsets Expecta-
tion-Maximization) on a 128 × 128 matrix (slice thickness, 5 mm), 2 iterations eight subsets, 3
mm FWHM Gaussian filter, and corrected by photon attenuation using the CT scan.
The image data analysis and residence time calculation were performed by the method
reported previously [45–47]. Each CT and PET whole-body image was successively loaded to
the PMOD 3.0 software (PMOD technologies, Switzerland; www.pmod.com) to generate the
fusion images. The regions of interest (ROIs) of these organs were manually drawn as precisely
as possible on the organ itself, on each horizontal slice. Organs with high uptake of [18F]T807,
such as brain, lungs, and heart were easily identified and outlined. Organs with low uptake of
[18F]T807, or those with complicated anatomy such as liver, gallbladder, kidneys, and bladder,
were processed manually. ROIs were adjusted (slice by slice) for organ movement (such as
intestine peristalsis) between time frames. In order to evaluate the significant tracer accumula-
tion in large intestine of mice, the ROI of the large intestine was defined as the sum of [18F]
T807 uptakes for all time points. The activity in each organ was non-decay-corrected and
expressed as standard uptake value (SUV) (n = 2, Mean for monkeys; n = 4, Mean S.D. for
mice).
Radiation dosimetry evaluation of the [18F]T807 injection from animal
images
To calculate the absorbed radiation dose of each organ as well as the effective dose, the injected
dose of the [18F]T807 injection and the non-decay-corrected TACs of all source organs of two
monkeys and four mice were individually entered into an Excel spreadsheet, and the data was
processed individually for each animal. The difference between the injected dose and the sum
of the whole-body radioactivity (assuming that there was no excretion) plus the radioactivities
of the above-mentioned organs was taken as the TAC of the remainder.
The residence time (h) of the selected organ was calculated as the area under the TACs of
the source organ from time zero to infinity over the initial total body activity (5 half-lives of
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GMP-compliant [18F]T807 injection for preclinical and clinical use
18F, i.e. approximately 550 min). The area under the TAC curve of each organ was generated
using the following strategy, including trapezoidal integration of the first four TAC data points
through the origin and exponential decline of the four remaining TAC data points to infinity
such that the residence times of these organs were obtained [45].
The human dosimetry was estimated from both mice and monkeys biodistribution data.
The organ weight and body mass were used for allometric scaling [47]. That is, the residence
time in each organ was converted to the corresponding human value by multiplication with a
factor to scale organ and body weights (in kg) as (wm,b/ wm,o)(wh,o/ wh,b), where wm,b was the
monkey body weight, wm,o was the monkey organ weight, wh,b was the human body weight,
and wh,o was the human organ weight. However, the wm,o values of Formosan rock monkeys
(Macaca cyclopis) were unavailable. We have instead used the wm,o values of rhesus monkey
(Macaca mulatta) in our calculations. Both monkeys’ organ weights [48, 49] and humans’
organ weights [50] were obtained from the literature. The residence time of [18F]T807 in the
rest of the body was obtained by subtracting the total organ residence time from the reciprocal
of the 18F decay constant.
Absorbed radiation doses were calculated from the residence times in all source organs for
each monkey by entering the information into the Java-based OLINDA 1.0/EXM computer
program [51] using the model for a 70-kg adult male and female phantom.
PET/CT imaging of the [18F]T807 injection in an AD patient
Subject. The representative subject (male, age = 61) in this study was noted to have visuo-
spatial problem and impaired memory function. In addition, he had completed more than 16
years of education and worked as a medical professional. His Mini-Mental State Exam
(MMSE) [52] was 24, which was mildly impaired for his education and occupation. The neuro-
psychological test showed impaired verbal (word sequence learning) and non-verbal memory
(Benton Visual Retention Test), impaired language function (object naming and semantic ver-
bal fluency), visual perceptual function (3D block deign), and executive function (Wisconsin
Card Sorting Test). Based on the NIA-AA workgroups in 2011 [53], the clinical diagnosis was
early onset AD, probably a posterior cortical atrophy variant.
PET imaging. Imaging with [18F]T807 was performed with GE Healthcare Discovery ST4
PET/CT scanner (2D mode, 47 image planes, 15.0 cm axial field of view). After 80–100 min of
intravenous injection of approximately 185 MBq (specific activity = 151 GBq/μmol) of [18F]
T807, PET images of human brain were acquired over 20 min and processed by the method
reported previously [12, 54].
Results and discussion
GMP production of the [18F]T807 injection
[18F]T807 (1) is a potent tau imaging agent and has been synthesized by several methods in
various radiochemical yields and radiochemical purities [11, 12, 38–40]. Initially, [18F]T807
was synthesized by nucleophilic fluorination of the nitro-precursor (T807P, 2) with [18F]F−,
followed by reduction with Fe/HCl and purification with HPLC, to give the desired product in
29–47% yield (EOB) (Fig 1) [12]. However, this synthesis sequence was somewhat compli-
cated. Thus, several improved methods have been reported [38–40]. Among these, Shoup et al.
have developed a new method to synthesize [18F]T807 by radiofluorination of its t-Boc pro-
tected nitro- precursor (tBoc-T807P, 3) with [18F]F- followed by on-line de-protection with
HCl/HPLC to give [18F]T807 in 16–25% (EOB) in a synthesis time of 60 min (Fig 2) [40]. We
have tried to adapt this method to synthesize [18F]T807 for our pre-clinical and clinical studies.
However, we encountered some difficulty in repeating this method. For example, when the
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 5. Representative semi-preparative HPLC chromatograms of [18F]T807 synthesized by Method A (on-line one-
step t-Boc deprotection and HPLC purification, Waters HSS T3, 5 μm, 10 × 250 mm, 18% EtOH(pH 2.0, adjusted by
HCl), 5.0 ml/min, 254 nm) (A) and Method B (two-step t-Boc deprotection and HPLC purification, Phenomenex
Luna(2) HILIC, 5 μm, 10 × 250 mm, 0.1% HCl(aq), 254 nm, 3 mL/min) (B).
https://doi.org/10.1371/journal.pone.0217384.g005
reaction mixture was injected into HPLC, we have found that the retention time (Rt) of [18F]
T807 chromatogram (Rt = 40~49 min, n = 3, as shown in Fig 5(A)) was significantly different
from that of the reported value (22~24 min). In addition, a significant amount of radioactivity
was lost during on-line de-protection with HCl/HPLC purification, SPE post-formulation and
sterile filtering, resulting in low overall RCYs of [18F]T807 (1~3%, EOS, n = 3) compared to
Shoup’s data (14 3%, EOS). Although using the on-line de-protection of intermediate ([18F]
4) and purification of final product could simplify the production process for the [18F]T807
injection, the reproducibility of this method is something to be concerned about. It has been
reported that this de-protection route may rely on several factors such as the strength and stoi-
chiometry of the acid used, and the flow rate of HPLC eluent, etc. [55]. As a result, we have
chosen to use HCl for in-situ de-protection of the intermediate followed by purification of
[18F]T807 with HPLC (Phenomenex Luna (2) HILIC column; 5 μm, 10 × 250 mm; eluent:
0.1% HCl(aq); flow rate: 3 ml/min). The semi-preparative HPLC chromatogram of [18F]T807
purified by this method was depicted in Fig 5(B). Using this modified purification method, we
are able to produce [18F]T807 routinely with a FXFN Module in 20.5 6.1% yield (EOB,
n = 15) in a synthesis time of 70 5 min from EOB. Typically, start with 27.4 16.6 GBq of
[18F]F−, 3.4 2.3 GBq of the [18F]T807 injection was produced at EOS. Both the chemical and
radiochemical purity of the [18F]T807 injection were >90% with a specific activity of 151 52
GBq/μmol (n = 15, EOS), and has been used for pre-clinical and clinical studies.
After the completion of this study, a similar synthetic method with a home-made module
was used to synthesize [18F]T807 in 20–30% yield (EOB) in a synthesis time of 60–70 min
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 6. Representative analytical radio-HPLC (A) and radio-TLC chromatograms (B) of [18F]T807 at 0, 2 and 4 hrs
after EOS.
https://doi.org/10.1371/journal.pone.0217384.g006
from EOB [38]. However, this synthetic method required solid phase extraction and reformu-
lation of [18F]T807 after HPLC purification. Most recently, a new t-Boc-protected precursor
(AV-1622) with trimethylammonium as a leaving group has been used to synthesize [18F]T807
in 25~55% yield (EOB) in a synthesis time of 45~60 min [56–60]. The applicability of this new
precursor for routine automated production of [18F]T807 has not yet been proven.
Quality control and stability test of the [18F]T807 injection
The QC test results of three batches of validation runs of the [18F]T807 injection are tabulated
in Table 1. The quality of the [18F]T807 injection synthesized by this method met the USP cri-
teria: The appearance of the [18F]T807 injection was clear, the HPLC retention time of the
[18F]T807 injection was approximately10 min (Fig 6(A)), the retardation factor (Rf) values of
[18F]F− and the [18F]T807 injection were approximately 0.0 and 0.4, respectively (Fig 6(B)), the
radiochemical purity of the [18F]T807 injection was greater than 90% (Fig 6), and the concen-
trations of residual solvents in the [18F]T807 injection were Acetone < 0.5%, DMSO < 0.5%,
EtOH < 0.5%, and ACN < 0.04% (Table 1). The stability test showed that the [18F]T807 injec-
tion was stable at room temperature for up to 4 h after EOS (Table 2).
Whole-body biodistribution of the [18F]T807 injection in mice and
monkeys
The [18F]T807 injection has been demonstrated as a potent tau imaging agent in humans [12,
61–63]. However, its radiation dosimetry in humans was not available until very recently [64].
Table 2. Stability of three consecutive productions of the [18F]T807 injection (n = 3).
Test
Acceptance Criteria
Elapsed time (hr)
Run 1
91.5
90.5
90.6
0.1
Run 2
92.38
91.90
91.73
0.26
Run 3
93.12
91.86
91.08
0.26
Radiochemical purity (%)
> 90%
0
2
4
0
2
4
Residual [18F]fluoride (%)
< 5%
1.4
1.16
0.58
2.5
1.95
0.66
https://doi.org/10.1371/journal.pone.0217384.t002
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 7. Representative whole-body biodistribution of [18F]T807 in normal mice at last time-frame post injection
and non-decay-corrected time-activity curves of [18F]T807 in various organs of non-fasted mice (n = 4) and fasted
mice (n = 3).
https://doi.org/10.1371/journal.pone.0217384.g007
In order to fulfill the Taiwanese Food and Drug Administration (TFDA) requirements, we
studied its organ radiation dose burden in animal models and extrapolated it to humans before
human PET studies are undertaken.
Ex vivo studies of [18F]T807 in mice showed that [18F]T807 cleared rapidly from the brain
and had high uptake in the kidneys, the bladder, and the liver, and relatively low uptake in
muscle and bone [11].
PET studies of the [18F]T807 injection in mice and monkeys showed that [18F]T807 did not
produce overt adverse effects, and its uptake in gallbladders of fasted and non-fasted mice var-
ied significantly (Fig 7). It also showed that the uptake of radioactivity in mice GB and LLI
were high and increased with time (Fig 7), while its uptake in the monkey’s lung, liver, and kid-
neys were high in the beginning and gradually declined thereafter (Fig 8). The radioactivity in
the gallbladder and bladder increased with time, suggesting that [18F]T807 may be eliminated
via hepatobilliary and renal systems, which was in agreement with Choi’s results [64].
Radiation dosimetry and toxicity of the [18F]T807 injection
The organ radiation absorbed dose studies of the [18F]T807 injection were carried out in both
fasted and non-fasted ICR mice and Formosan rock monkeys. The residence times of selected
organs in mice from the TACs of both the ex vivo biodistribution studies [11] and the PET
imaging are tabulated in Table 3.
Using these residence times, the individual organ doses in humans as extrapolated from the
mice are tabulated in Table 4 [64]. The results from microPET imaging showed that the target
organs of the [18F]T807 injection were the gallbladder, LLI, and bladder, and the estimated
effective doses extrapolated from fasted and non-fasted mice were 123.0 27.4 μSv/MBq
(n = 3) and 93.7 19.0 μSv/MBq (n = 4), respectively. In contrast, the results from ex vivo stud-
ies, which include only kidneys, liver, brain, muscle, and bone [11], showed that the estimated
effective dose was approximately 14 μSv/MBq (Table 4). The exact reason(s) for the discrep-
ancy between ex vivo and in vivo studies is unclear, but omission of some mice organs in ex
vivo studies may account for this difference. Although partial volume effects in microPET
imaging caused by the very small mice organ size may contribute to the underestimation of
the estimated effective dose, microPET imaging is still regarded as a useful tool for absorbed
dose estimations in humans.
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 8. Typical sagittal view of [18F]T807 whole-body images of a male Formosan Rock monkey at different time points
(A) and Non-decay-corrected time-activity curves of [18F]T807 in various organs of monkeys (n = 2) (B).
https://doi.org/10.1371/journal.pone.0217384.g008
To estimate the human dosimetry from the Formosan rock monkey whole-body PET
images data, the organ weight and body mass were used for allometric scaling after obtaining
the residence time of each organ for each subject. However, the organ weights of Formosan
rock monkey are unavailable; since rhesus monkeys (Macaca mulatta) are genetically similar
to Formosan rock monkeys (Macaca cyclopis) [65, 66], we used organ weights of rhesus mon-
keys to calculate human dosimetry. The results (Table 4) showed that the human estimated
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Table 3. Residence times (hr) of selected organs from the biodistribution data and microPET imaging of mice and monkeys.
Source Organ
Residence times (hr)
Ex Vivo Data
Mice
In Vivo Imaging
Fasted Mice
(n = 3)
Non-fasted Mice
(n = 4)
Fasted Monkey
(n = 2)
Brain
Lung
0.011
0.011 0.003
0.044 0.009
0.003 0.001
0.025 0.004
-
0.024 0.008
0.056 0.030
0.003 0.001
0.033 0.017
-
0.023 0.008
0.111 0.027
0.035 0.002
0.199 0.134
-
-
Heart
-
0.046
0.013
0.005
-
Liver
Muscle
Bone
-
-
-
Spleen
GB
-
-
0.005 0.002
0.477 0.031
0.178 0.030
0.045 0.004
0.110 0.008
1.457 0.150
-
0.251 0.212
1.373 0.354
0.036 0.034
0.352 0.167
0.546 0.569
0.525 0.387
1.075 0.322
0.029 0.026
0.148 0.087
0.747 0.659
LLI
-
Kidneys
Bladder
Remainder
0.019
-
2.547
https://doi.org/10.1371/journal.pone.0217384.t003
Table 4. Radiation dosimetry estimates of the [18F]T807 injection extrapolated from the mice and monkey biodistribution data.
Target Organ
Radiation Dosimetry (μGy/MBq)
In Vivo Imaging
Ex Vivo Data
Mice
Fasted
Mice
(n = 3)
Non-Fasted Mice
(n = 4)
Fasted
Monkey
(n = 2)
Human [64]
Adrenals
Brain
14.7
4.6
7.1 2.0
2.4 1.2
9.7 0.5
5.3 1.5
16.3 0.7
5.5 1.5
18.7
12.7
9.8
Breasts
12.2
15.7
16.0
17.6
15.9
17.0
16.2
18.6
11.2
13.2
7.4
3.0 2.0
4.7 2.0
7.7 0.2
Gallbladder
LLI wall
399.4 330.5
814.7 204.5
31.4 4.7
10.0 0.9
20.4 3.1
5.0 2.2
632.4 635.1
626.3 153.1
28.5 4.7
11.8 0.0
21.9 5.7
7.2 1.8
763.5 46.0
11.7 0.2
19.2 2.2
14.0 0.2
66.3 40.0
18.9 0.4
71.4 48.9
38.8 15.6
24.8 4.6
10.0 0.4
14.1 0.1
20.6 0.7
10.1 0.3
12.9 3.3
8.3 0.6
44.9
13.0
31.6
13.6
16.8
24.6
34.9
81.2
49.5
10.8
14.2
17.7
18.4
20.5
7.8
Small Intestine
Stomach
ULI wall
Heart wall
Kidneys
29.2 19.3
10.6 3.5
9.6 2.5
25.9 11.1
14.3 8.9
12.4 3.7
9.6 1.1
Liver
Lungs
Muscle
9.2 0.9
Ovaries
16.3
16.3
12.3
21.9
9.9
53.0 9.5
10.2 1.3
12.1 0.6
8.4 3.2
42.3 5.7
14.3 3.6
12.0 0.6
10.5 3.7
5.4 1.8
Pancreas
Red marrow
Osteogenic Cells
Skin
4.4 1.5
Spleen
15.3
12.8
13.7
12.8
15.6
6.9 2.4
8.6 2.0
12.9 3.3
7.9 0.1
12.6
8.4
Testes
11.8 0.3
3.4 2.7
10.2 2.0
5.4 2.7
Thymus
8.3 0.6
11.8
9.4
Thyroid
3.0 2.8
5.0 3.1
7.8 0.7
Urinary bladder
185.6 78.0
74.1 41.9
61.3 3.3
42.0
(Continued)
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Table 4. (Continued)
Target Organ
Radiation Dosimetry (μGy/MBq)
In Vivo Imaging
Non-Fasted Mice
Ex Vivo Data
Mice
Fasted
Mice
(n = 3)
Fasted
Monkey
(n = 2)
Human [64]
(n = 4)
Uterus
16.9
12.4
31.7 4.7
11.0 0.6
23.5 1.4
11.3 0.6
μSv/MBq
98.0 46.8
93.7 19.0
14.9 0.1
11.5 0.0
14.7
-
Total body
Effective dose equivalent (EDE)
Effective dose (ED)
15.2
13.8
105.3 32.9
123.0 27.4
69.5 1.4
18.9 1.4
-
22.5
https://doi.org/10.1371/journal.pone.0217384.t004
dosimetry of [18F]T807 in most organs ranged between 6 and 764 μGy/MBq. The critical
organ of [18F]T807 was the gallbladder. The estimated effective dose of [18F]T807 extrapolated
from monkeys was 19 μSv/MBq (n = 2), which was in agreement with human data from Choi’s
study [64] and was comparable to those of many 18F-labeled PET tracers [67, 68]. The guide-
lines on radiation exposure for human subjects involved in research studies varied internation-
ally. Based on the guidelines of National Institutes of Health (NIH), the maximum exposure is
50 mSv of effective dose per year for a research subject [69]. On the other hand, under the
guidelines of the European commission, the intermediate risk levels in adults will be equivalent
to an effective dose range of 1–10 mSv per annum [69].
The toxicity of T807 has not been reported. However, the amounts of T807 that have been
injected into the subjects in previous [18F]T807 human studies have been reported to be
0.2~0.6 μg (Table 5) [12, 34, 64, 70–75]. Thus, the amount of [18F]T807 (185 18.5 MBq
or � 0.32 μg of T807) that will be injected to the subjects of our clinical study is reasonable and
within the safety range.
PET/CT imaging of the [18F]T807 injection in an AD patient
Since the GMP-compliant [18F]T807 injection synthesized by this one-pot two-step method
seems to be safe for human study, we carried out the PET/CT Imaging of the [18F]T807 injec-
tion in an AD patient (Fig 9). The results showed that [18F]T807 did not produce overt adverse
Table 5. Safety summary of the [18F]T807 injection.
Reference
Subject type
Subject
number
Activity injected
(MBq)
Specific Activity (GBq/μmol,
EOS)
Amount of non-radioactive T-807
injected (μg)
< 2.63
Safety
Note
[12]
3HC, 1 MCI, 2 AD
1 HC
6
370
37
Yes
[70]
[34]
1
190
222 52
216 60
0.22
Yes
-
56 HC, 13 MCI, 6AD 19
MCI/AD
75
333~407
< 0.50
[64]
[71]
[72]
[73]
[74]
[75]
6 HC
10 HC, 6 PSP, 6PD
8 HC, 8 AD
5 HC, 5 AD
21 HC, 3 MCI
59 HC, 61 AD/MCI
Total
6
22
16
10
24
120
280
1
268 31
175~183
303~341
225 16
303~341
280 37
175~407
185
342 192
240–560
> 200
0.21
< 0.20
< 0.44
< 0.63
< 0.41
< 0.29
0.20~0.63
0.32 μg
Yes
-
-
93~272
216 60
> 250
-
-
-
37~560
151
-
This Study
AD
Yes
https://doi.org/10.1371/journal.pone.0217384.t005
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GMP-compliant [18F]T807 injection for preclinical and clinical use
Fig 9. Representative axial PET images of [18F]T807 of an AD patient (male, age = 61, MMSE = 24).
https://doi.org/10.1371/journal.pone.0217384.g009
effects clinically, and had considerable uptake in the lateral temporal lobe, mesial temporal
lobe, hippocampal area, and occipital lobe, which is consistent with the results reported in the
literature [33].
Conclusions
With this one-pot two-step synthesis of the [18F]T807 injection using a FXFN module, a GMP-
compliant [18F]T807 injection can be automatically produced with high reproducibility and
high quality. PET imaging and radiation dosimetry evaluation in mice and Formosan rock
monkeys suggested that the [18F]T807 injection synthesized by this method is suitable for use
in human PET imaging studies. Preliminary study in an AD patient showed that [18F]T807
bound to tau protein, and clinical studies are in progress for imaging tauopathies in humans.
Supporting information
S1 Fig. The identity of t-Boc protected precursor (3) of [18F]T807 was confirmed by
1H-NMR. 1H-NMR (400 MHz, CDCl3): δ 1.80 (s, 9H),7.71 (dd, J = 8.0, 1.6 Hz, 1H), 8.15 (d,
J = 5.8 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 8.33 (dd,J = 8.5, 2.3 Hz,1H), 8.40 (d, J = 8.0 Hz, 1H),
8.70 (d, J = 5.8Hz, 2H), 8.97 (d, J = 2.0Hz, 1H), 9.3 (d, J = 0.6Hz, 1H).
(TIF)
S2 Fig. The identity of t-Boc protected precursor (3) of [18F]T807 was confirmed by
13C-NMR. 13C-NMR (100MHz, CDCl3): δ 28.5, 86.0, 111.3, 115.87, 118.0, 121.2, 121.3, 123.4,
124.9, 135.2, 138.2, 139.3, 142.8, 143.2, 144.1, 147.5, 148.7, 150.4, 155.9.
(TIF)
S3 Fig. The identity of t-Boc protected precursor (3) of [18F]T807 was confirmed by mass
spectra. ESIHRMS: Calcd for C21H18N4O4Na [M+Na]+, 413.1220; found, 413.1221.
(TIF)
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GMP-compliant [18F]T807 injection for preclinical and clinical use
S4 Fig. The chemical purity of 3 was determined by RP-HPLC. RP-HPLC (Phenomenex
Gemini C18, 5μm, 4.6×250 mm, MeCN/0.05M NH4OAc (7/3), 254nm, 1mL/min, 10μl)
(TIF)
Acknowledgments
We are grateful to Mr. Pei-Yao Lin, Mr. Kuang-Hua Tang and Mr. Wei-Hua Kuo of the PET
Center, Department of Nuclear Medicine, National Taiwan University Hospital and Mr. Dai-
Kai Chou and Dai-Wei Tzeng of the PET Center, Department of Nuclear Medicine, Tri-Ser-
vice General Hospital for their excellent technical assistances.
Author Contributions
Conceptualization: Ya-Yao Huang, Chyng-Yann Shiue.
Data curation: Ya-Yao Huang, Chia-Ling Tsai, Hao-Yu Hsieh, Ching-Hung Chiu, Ling-Wei
Hsin.
Formal analysis: Ming-Jang Chiu, Ruoh-Fang Yen, Chi-Han Wu.
Funding acquisition: Ming-Jang Chiu, Ruoh-Fang Yen, Kai-Yuan Tzen, Cheng-Yi Cheng.
Investigation: Ya-Yao Huang, Ming-Jang Chiu, Ruoh-Fang Yen, Chia-Ling Tsai, Hao-Yu
Hsieh, Ching-Hung Chiu, Chi-Han Wu, Ling-Wei Hsin, Cheng-Yi Cheng, Kuo-Hsing Ma.
Methodology: Ya-Yao Huang, Ming-Jang Chiu, Chia-Ling Tsai, Hao-Yu Hsieh, Ling-Wei
Hsin, Kuo-Hsing Ma.
Project administration: Chia-Ling Tsai, Ching-Hung Chiu, Chi-Han Wu, Ling-Wei Hsin,
Kai-Yuan Tzen, Chyng-Yann Shiue.
Resources: Ming-Jang Chiu, Ruoh-Fang Yen, Ling-Wei Hsin, Kai-Yuan Tzen, Cheng-Yi
Cheng, Kuo-Hsing Ma.
Supervision: Ya-Yao Huang, Kai-Yuan Tzen, Kuo-Hsing Ma, Chyng-Yann Shiue.
Validation: Ya-Yao Huang, Ching-Hung Chiu.
Visualization: Chyng-Yann Shiue.
Writing – original draft: Ya-Yao Huang.
Writing – review & editing: Ya-Yao Huang, Chyng-Yann Shiue.
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