Blood–brain barrier permeability of ginkgolide: Comparison of the behavior of PET probes 7α-[18F]fluoro- and 10-O-p-[11C]methylbenzyl ginkgolide B in monkey and rat brains

Article abstract of DOI:10.1016/j.bmc.2016.08.032

The blood–brain barrier permeability of ginkgolide B was examined using positron emission tomography (PET) probes of a¹⁸F-incorporated ginkgolide B ([¹⁸F]-2) and a¹¹C-incorporated methylbenzyl-substituted ginkgolide B ([¹¹C]-3). PET studies in monkeys showed low uptake of [¹⁸F]-2 into the brain, but small amounts of [¹¹C]-3 were accumulated in the parenchyma. Furthermore, when cyclosporine A was preadministered to rats, the accumulation of [¹⁸F]-2 in the rat brain did not significantly change, however, the accumulation of [¹¹C]-3 was five times higher than that in the control rat. These results provide effective approaches for investigating the drug potential of ginkgolides.

Full text of DOI:10.1016/j.bmc.2016.08.032

Bioorganic & Medicinal Chemistry 24 (2016) 5148–5157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Blood–brain barrier permeability of ginkgolide: Comparison of the
behavior of PET probes 7a
-[18F]fluoro- and 10-O-p-[11C]methylbenzyl
ginkgolide B in monkey and rat brains
Hisashi Doi a, Kengo Sato b, Hideo Shindou c, Kengo Sumi d, Hiroko Koyama d, Takamitsu Hosoya e,
Yasuyoshi Watanabe a, Satoshi Ishii f, Hideo Tsukada g, , Koji Nakanishi h, Masaaki Suzuki i,
⇑
⇑
a Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies (CLST), 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
b PET Medical Application Group, Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu 434-8601, Japan
c Department of Lipid Signaling, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
d Division of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
e Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
f Department of Immunology, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
g PET Center, Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu 434-8601, Japan
h Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
i Department of Clinical and Experimental Neuroimaging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The blood–brain barrier permeability of ginkgolide B was examined using positron emission tomography
(PET) probes of a 18F-incorporated ginkgolide B ([18F]-2) and a 11C-incorporated methylbenzyl-substi-
tuted ginkgolide B ([11C]-3). PET studies in monkeys showed low uptake of [18F]-2 into the brain, but
small amounts of [11C]-3 were accumulated in the parenchyma. Furthermore, when cyclosporine A
was preadministered to rats, the accumulation of [18F]-2 in the rat brain did not significantly change,
however, the accumulation of [11C]-3 was five times higher than that in the control rat. These results pro-
vide effective approaches for investigating the drug potential of ginkgolides.
Received 9 June 2016
Revised 18 August 2016
Accepted 19 August 2016
Available online 23 August 2016
Keywords:
Ginkgolide B
Ó 2016 Elsevier Ltd. All rights reserved.
Platelet-activating factor
Blood–brain barrier
PET
C-[11C]methylation
1. Introduction
treatment of cerebral insufficiencies, including Alzheimer’s dis-
ease.1–6 G. biloba extracts are subdivided into two major groups,
Ginkgo biloba (G. biloba) extracts have been shown to have neu-
roprotective effects both in vivo and in vitro.1–6 In some parts of
Asia and in Western countries, the standardized G. biloba extracts
(e.g., EGb 761) have been utilized as complementary medicines
for the improvement of peripheral and cerebral blood flow, and
the flavone glycosides and the terpene lactones, which are thought
to contribute to its vasotropic and neuroprotective effects. The
most attractive components of the extract are ginkgolides (terpene
lactones), which are believed to play an important role in the neu-
romodulatory effects of G. biloba. They have been shown to act as
platelet-activating factor (PAF) receptor antagonists,7–9 glycine
receptor antagonists,10–14 GABAA receptor antagonists,15,16 5-HT3
receptor antagonists,17,18 and inhibitors of neuronal cell death
caused by b-amyloid aggregation.19–22 However, the molecular
mechanisms underlying the action of ginkgolides in the central
nervous system (CNS) are poorly understood. In particular, deter-
mining whether ginkgolides can penetrate the blood–brain barrier
(BBB) has been a challenge.23 In this regard, Nanjing group investi-
gated the BBB permeability of ginkgolide B by an LC-MS/MS
method.23 On the other hand, we speculated that in vivo imaging
of ginkgolides, using nuclear medicine techniques such as positron
Abbreviations: BBB, blood–brain barrier; CNS, central nervous system; cDNA,
complementary DNA; CsA, cyclosporine A; MRI, magnetic resonance imaging;
MDR1, multidrug resistance 1; PAF, platelet-activating factor; PET, positron
emission tomography; ROIs, regions of interest; SA, specific activity; SUV,
standardized uptake value.
⇑
Corresponding authors. Tel.: +81 53 586 7111 (H.T.), +81 562 46 2311 (M.S.).
E-mail addresses: hisashi.doi@riken.jp (H. Doi), satoken@crl.hpk.co.jp (K. Sato),
hshindou-tky@umin.net (H. Shindou), kesumi@dwti.co.jp (K. Sumi), hirokok@
gifu-u.ac.jp (H. Koyama), thosoya.cb@tmd.ac.jp (T. Hosoya), yywata@riken.jp
(Y. Watanabe), satishii@med.akita-u.ac.jp (S. Ishii), tsukada@crl.hpk.co.jp
(H. Tsukada), kn5@columbia.edu (K. Nakanishi), suzukims@ncgg.go.jp (M. Suzuki).
http://dx.doi.org/10.1016/j.bmc.2016.08.032
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
5149
emission tomography (PET), would provide valuable information
and insights into the BBB permeability of these compounds, from
the viewpoint of molecular biology. Here, we studied the BBB per-
meability in both rat and monkey brains using two PET probes
The collected PET data were reconstructed by using the filter back
projection method with a Hanning 4.5 mm filter. Quantitative
analyses of the PET data were performed by Patlak plot analysis,27
using PMOD software (PMOD Technologies, Zurich, Switzerland).
Regions of interest (ROIs) were defined on the monkey PET images
and superimposed on magnetic resonance imaging (MRI) record-
ings, which were simultaneously obtained, to validate the accuracy
of the location.28
derived from ginkgolide
B
(1),
7a
-[18F]fluorine-substituted
ginkgolide B ([18F]-2) and 10-O-p-[11C]methylbenzyl ginkgolide
B ([11C]-3), the latter of which was efficiently synthesized using
our rapid C-[11C]methylation methods.24,25
The animal experiments were approved and performed in
accordance with the guidelines of the Central Research Laboratory,
Hamamatsu Photonics.
2. Materials and methods
2.1. General methods
1H and 13C nuclear magnetic resonance (NMR) spectra were
2.2. PAF receptor binding assay of ginkgolide B (1), 10-O-p-
methylbenzyl ginkgolide
ginkgolide B (4); Figure 1
B (3), and 10-O-p-bromobenzyl
recorded on a JEOL JNM
a-400 or a JNM ECS-400 spectrometer.
The proton chemical shifts are expressed in parts per million
(ppm) downfield from tetramethylsilane (d 0.00 and d 0.00) or in
ppm relative to CDCl3 (d 7.24 and d 77.0) in 1H and 13C NMR, respec-
tively. The abbreviations s, d, t, q, and m signify singlet, doublet, tri-
plet, quartet, and multiplet, respectively. High-resolution mass
spectra (HRMS) were measured on JEOL JMS-700/GI. EGb 761Ò
was supplied by Dr. Willmar Schwabe Pharmaceuticals. [3H]WEB
2086 was purchased from NEN Life Science Products (Boston, MA).
The other chemicals and solvents were purchased from Sigma–
Aldrich Japan (Tokyo, Japan), Wako Pure Chemical Industries
(Osaka, Japan), Tokyo Kasei Kogyo (Tokyo, Japan), Nacalai Tesque
(Kyoto, Japan), and Kanto Kagaku (Tokyo, Japan), and were used
without further purification. Silica gel column chromatography
Based on a previously reported method,29 the PAF receptor
binding assays of ginkgolide B (1), 10-O-p-methylbenzyl ginkgolide
B (3), and 10-O-p-bromobenzyl ginkgolide B (4) were performed
using [3H]WEB 2086 as a radioligand for the PAF receptor antago-
nist. In this experiment, membrane fractions from hearts and
skeletal muscles of PAF receptor-transgenic mice (157.5 fmol of
PAF receptor) were mixed with a 10 nM buffer solution containing
[3H]WEB 2086. The compounds 1, 3, and 4 were tested in triplicate
using a 96-well microplate.
2.3. Cell permeability analyses of ginkgolide
fluorinated ginkgolide (2), and 10-O-p-methylbenzyl
ginkgolide B (3) using a P-glycoprotein-expressing cellular
membrane; Table 1
B (1), 7a-
was performed using Merck SiliaFlashÒ F60 (40–63
lm).
B
[
11C]CO2 was produced by a 14N(p, 11C nuclear reaction using
a)
a cyclotron (HM-18, Sumitomo Heavy Industry, Tokyo, Japan) at
the Hamamatsu Photonics PET Center. [11C]CH3I was prepared by
reducing [11C]CO2 with lithium aluminum hydride, followed by
iodination with hydriodic acid using a modified CUPID system as
a remote-control synthesizer (Sumitomo Heavy Industry, Tokyo,
Japan), consisting of systems for heating the reaction mixture, dilu-
tion, high performance liquid chromatography (HPLC) injection,
fractional collection, evaporation, and sterile filtration. Purification
with a semi-preparative HPLC was performed on a JASCO system
(Tokyo, Japan). Radioactivity was quantified with an IGC-7 dose
calibrator (Hitachi Aloka Medical, Tokyo, Japan). An analytical
Cell permeability analyses of compound 1, 7a-fluorinated gink-
golide B (2), and compound 3 were performed based on previously
reported methods.30,31 Control cells (LLC-PK1 cells) and P-glyco-
protein-expressing cells (LLC-PK1 cells transfected with human
MDR1 cDNA) were seeded on a cell culture insert (Falcon 3-
353096, material: PET, pore size: 3.0 l
m, growth area: 0.3 cm2)
and allowed to form a confluent monolayer (8 days) prior to the
permeability experiment.
HPLC was performed on
a
Shimadzu system (Kyoto, Japan)
2.4. Synthesis of 10-O-p-methylbenzyl ginkgolide B (3) and 10-
O-p-bromobenzyl ginkgolide B (4)
equipped with pumps and a UV detector, and the effluent radioac-
tivity was measured using a Gamma Detector (FGD-102, Hitachi
Aloka Medical, Tokyo, Japan). To image the monkey brain, we used
a Hamamatsu SHR-7700 as a high-resolution animal PET scanner.26
Compounds 3 and 4 were synthesized according to a previously
reported method.32

5150
H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
Characterization data for compound 3: 1H NMR (400 MHz,
CDCl3): d 1.13 (9H, s, t-Bu), 1.29 (3H, d, J = 6.8 Hz, H-16), 1.89–
1.91 (2H, m, H-7 , H-8), 2.26 (1H, d, J = 8.2 Hz, H-7b), 2.35 (3H, s,
(3C), 13.7 (3C), 27.4 (3C), 29.1 (3C), 29.3 (3C), 32.3, 37.1, 41.7,
49.1, 67.8, 72.7, 74.3, 75.9, 76.8, 79.9, 83.6, 90.7, 98.6, 110.3,
128.1 (2C), 134.1, 137.5 (2C), 144.6, 171.0, 171.3, 175.4; HRMS
(ESI) m/z: [M+H]+ calculated for C39H57O10119Sn, 805.2977; exper-
imental 805.2973.
a
CH3), 2.80–2.82 (1H, broad singlet, OH-3), 2.83 (1H, d, J = 3.2 Hz,
OH-1), 3.04 (1H, q, J = 6.8 Hz, H-14), 4.25 (1H, dd, J = 8.0, 3.2 Hz,
H-1), 4.52 (1H, d, J = 8.0 Hz, H-2), 4.54 (1H, d, J = 9.6 Hz, CH2),
4.89 (1H, s, H-10), 5.29–5.30 (1H, m, H-6), 5.44 (1H, d, J = 9.6 Hz,
CH2), 5.97 (1H, s, H-12), 7.19–7.25 (4H, m, aromatic); 13C NMR
(100 MHz, CDCl3): d = 7.4, 21.4, 29.3 (3C), 32.3, 37.1, 41.7, 49.0,
67.9, 72.7, 73.9, 74.3, 75.8, 79.9, 83.6, 90.6, 98.6, 110.3, 130.0
(2C), 130.1 (2C), 131.7, 139.8, 171.1, 171.3, 175.5; HRMS (EI) m/z:
M+ calculated for C28H32O10, 528.1995; experimental, 528.1977.
Characterization data for compound 4: 1H NMR (400 MHz,
CDCl3): d 1.12 (9H, s, t-Bu), 1.27 (3H, d, J = 5.5 Hz, H-16), 1.87–
2.6. Synthesis of 10-O-p-[11C]methylbenzyl ginkgolide B ([11C]-
3); Scheme 1, Figures 2 and 3
[
11C]CH3I (30–35 GBq) with a He gas stream (30 mL/min) was
trapped in a Pd2(dba)3 (1.8 mg, 1.97 mol) and P(o-CH3C6H4)3
(2.4 mg, 7.9 mol) solution in DMF (270 L) at room temperature.
The radioactive solution was transferred to a solution containing
the stannyl precursor 6 (3.6 mg, 4.5 mol), CuCl (2.0 mg, 20 mol),
and K2CO3 (2.8 mg, 20 mol) in DMF (60 L). Then, the mixture
was heated for 5 min at 65°C with hot air. After the addition of a
DMF:H2O (300 L; 1:5) solution, the mixture was injected into a
l
l
l
l
l
1.92 (2H, m, H-7
a, H-8), 2.27 (1H, d, J = 9.6 Hz, H-7b), 2.73 (1H,
l
l
d, J = 3.6 Hz, OH-1), 2.95 (1H, s, OH-3), 3.03 (1H, q, J = 6.8 Hz, H-
14), 4.24 (1H, dd, J = 8.3, 3.7 Hz, H-1), 4.53 (1H, d, J = 8.3 Hz, H-2),
4.54 (1H, d, J = 10 Hz, CH2), 4.89 (1H, s, H-10), 5.30 (1H, d,
J = 3.7 Hz, H-6), 5.45 (1H, d, J = 10 Hz, CH2), 5.98 (1H, s, H-12),
7.22 (2H, d, J = 8.2 Hz, aromatic), 7.54 (2H, d, J = 8.2 Hz, aromatic);
13C NMR (100 MHz, CDCl3): d = 7.4, 29.3 (3C), 32.3, 37.2, 41.7, 49.0,
67.8, 72.6, 73.4, 74.3, 76.1, 79.8, 83.6, 90.6, 95.9, 98.7, 110.3, 130.7
(2C), 134.2, 138.7 (2C), 171.0, 171.2, 175.6; HRMS (EI) m/z: M+ cal-
culated for C27H29BrO10, 592.0944; experimental, 592.0951.
l
semi-preparative HPLC system (mobile phase: CH3CN/
H2O = 57:43; column: Inertsil, ODS-3 10 mm internal diame-
ter ꢀ 250 mm,
5 lm; flow rate: 6.0 mL/min; UV detection:
220 nm; retention time: 16.5 min). The fraction of interest was col-
lected in a flask and concentrated in vacuo. In accordance to the
radiopharmaceutical formulation, 10-O-p-[11C]methylbenzyl gink-
golide B ([11C]-3) was dissolved in a mixed solution (4.1 mL) con-
taining intralipid (20% emulsion), saline, propylene glycol,
ethanol, and Tween 80 (volume ratio = 41:36:3:1:1), to then be col-
lected in a sterilized vial. The pH range and final sample volume
were 6.5–7.0 and 4.0 mL, respectively. The total synthesis time
was 34–40 min. [11C]-3 was identified by HPLC analysis using a
co-injection of the unlabeled reference compound 3. The radioac-
tivity of isolated [11C]-3 was 2.3–3.3 GBq. The [11C]CH3I-based
decay-corrected radiochemical yield was 30–40%. The radiochem-
ical and chemical purity were 95–98% and >99%, respectively. The
2.5. Synthesis of 10-O-p-iodobenzyl ginkgolide B (5) and 10-O-p-
(tri-n-butylstannyl)benzyl ginkgolide B (6)
10-O-p-Iodobenzyl ginkgolide B (5) was synthesized according
to a previously reported method.32
Characterization data for compound 5, 1H NMR (400 MHz,
CDCl3): d 1.12 (9H, s, t-Bu), 1.28 (3H, d, J = 6.8 Hz, H-16), 1.83–
1.95 (2H, m, H-7a, H-8), 2.27 (1H, d, J = 9.6 Hz, H-7b), 2.76 (1H,
d, J = 3.2 Hz, OH-1), 3.10 (1H, s, OH-3), 3.02 (1H, q, J = 6.8 Hz, H-
14), 4.24 (1H, dd, J = 8.4, 3.2 Hz, H-1), 4.52 (1H, d, J = 9.6 Hz, CH2),
4.53 (1H, d, J = 8.4 Hz, H-2), 4.89 (1H, s, H-10), 5.30 (1H, d,
J = 3.6 Hz, H-6), 5.43 (1H, d, J = 9.6 Hz, CH2), 5.98 (1H, s, H-12),
7.08 (2H, d, J = 8.8 Hz, aromatic), 7.74 (2H, d, J = 8.8 Hz, aromatic);
13C NMR (100 MHz, CDCl3): d = 7.4, 29.3 (3C), 32.3, 37.2, 41.7, 49.0,
67.8, 72.6, 73.4, 74.3, 76.1, 79.8, 83.6, 90.6, 95.9, 98.7, 110.3, 130.8
(2C), 134.2, 138.7 (2C), 171.0, 171.2, 175.6; HRMS (EI) m/z: M+ cal-
culated for C27H29IO10, 640.0805; experimental, 640.0831.
specific activity (SA) was calculated to be 12–45 GBq/lmol.
2.7. Monkey PET studies using the
7a
-[18F]fluorinated
ginkgolide B ([18F]-2) and [11C]-3 PET probes; Figures 4 and 5
Two young male monkeys (rhesus macaque) were subjected to
PET scans using 7
[
a
-[18F]fluorinated ginkgolide B ([18F]-2) and
11C]-3 probes. To minimize the effect of anesthetics, conscious
monkeys were used in this study. For each PET scan, the monkey
was seated on a purpose-designed chair and placed at a fixed posi-
tion in the PET gantry, with stereotactic coordinates aligned paral-
lel to the orbitomeatal line. After intravenous injection of the PET
The iodine compound 5 (40.4 mg, 63.1
(7.3 mg, 6.3 mol) were added to hexabutylditin solution
(96.0 L, 110 mg, 189 mol) in anhydrous DME (2.5 mL) at room
lmol) and Pd(PPh3)4
l
a
l
l
temperature. The solution was then refluxed for 12 h. After cooling,
20% KF (aq; 2 mL) was added. Then, the solution was filtered
through a Celite pad, and the pad was washed with hexanes. The
filtrate was extracted three times with ethyl acetate (5 mL each).
The combined organic layers were washed with brine (20 mL),
dried over Na2SO4, to then be filtered and evaporated. The residue
was purified by silica gel column chromatography (2 g silica gel;
hexane/ethyl acetate = 4:1 and 3:1) to obtain the colorless solid
probes ([18F]-2: 900 MBq, 50–140 GBq/
900 MBq, 12–45 GBq/
scan was performed for 120 min with [18F]-2 and for 64 min with
11C]-3. PET images of [18F]-2 and [11C]-3 were generated by the
lmol, 1.0 mL;
[
11C]-3:
l
mol, 1.0 mL) into each monkey, each PET
[
summation of all the image data that was collected at 34–64 min
after injection of the PET probe.
2.8. Common protocols for rat experiments
product, 10-O-p-(tri-n-butylstannyl)benzyl ginkgolide
B
(6)
(11.2 mg, 13.9
l
mol, 22% yield).
Male Sprague–Dawley rats (age, 7 weeks; weight, 210–235 g)
were used for the tissue dissection assay with [18F]-2, [11C]-3,
and [11C]verapamil ([11C]-7). Initially, rats were anesthetized with
choral hydrate (400 mg/kg, intraperitoneal injection). Subse-
quently, cyclosporine A (CsA; 10, 25, and 40 mg/kg) was adminis-
tered to rats 30 min prior to administering each PET probe; rats
in the control group were not treated with CsA. The target PET
Characterization data for compound 6, 1H NMR (400 MHz,
CDCl3): d 0.87 (9H, t, J = 7.3 Hz, CH3), 1.03–1.07 (6H, m, CH2),
1.12 (9H, s, t-Bu), 1.24–1.36 (6H, m, CH2), 1.31 (3H, d, J = 6.8 Hz,
H-16), 1.42–1.58 (6H, m, CH2), 1.93–1.99 (2H, m, H-8, 7a), 2.27
(1H, d, J = 8.7 Hz, H-7b), 2.83 (1H, s, OH), 2.87 (1H, d, J = 3.2 Hz,
H-10), 3.05 (1H, q, J = 6.8 Hz, H-14), 4.27 (1H, dd, J = 8.2, 3.2 Hz,
CH2), 4.53 (1H, d, J = 8.2 Hz, CH2), 4.58 (1H, d, J = 9.6 Hz, H-1),
4.89 (1H, s, OH), 5.32–5.33 (1H, m, H-6), 5.46 (1H, d, J = 9.6 Hz,
H-2), 5.98 (1H, s, H-12), 7.27 (2H, d, J = 7.8 Hz, aromatic), 7.49
(2H, d, J = 7.8 Hz, aromatic); 13C NMR (100 MHz, CDCl3): d 7.3, 9.7
probes,
(20 MBq, 12–45 GBq/
[
18F]-2 (5 MBq, 50–140 GBq/
mol, 0.5 mL), and
mol, 0.5 mL) were injected into each rat through the
l
mol, 0.5 mL),
[
11C]-3
l
[
11C]-7 (20 MBq,
37–55 GBq/
l
tail vein.

H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
5151
2.8.1. Tissue dissection assay of rats injected with [18F]-2, [11C]-
3, and [11C]-7; Figure 6
Rats were sacrificed by decapitation at 5, 15, 30, and 60 min
after the injection of the PET probe. Samples of blood, muscle,
and brain were rapidly removed, and the plasma was separated
from the blood sample by centrifugation. The weight and radioac-
tivity of each sample was measured with a gamma counter to then
calculate the standardized uptake values (SUVs).y Experiments
were repeated four times; the results are presented as mean stan-
dard deviation (SD).
2.8.2. Brain–blood perfusion to measure the remaining
radioactivity in the rat brain; Figure 7
At 30 min after the injection of [18F]-2 or [11C]-3, rats under-
went transcardial perfusion with a 10% formalin neutral buffer
solution (pH 7.4), followed by a saline perfusion. Then, the brains
were removed and the remaining radioactivity was measured.
The experiments were repeated at least two times; the results
are presented as mean SD.
Figure 1. Concentration–displacement curves for compounds 1, 3, and
4 as
measured by their ability to displace the binding of [3H]WEB 2086 to the cloned
PAF receptor.
2.8.3. Statistical analysis
3.2. Synthesis of radiolabeled ginkgolide probes, [18F]-2 and
Statistical analysis was performed with SPSS (Windows soft-
ware, version 21, SPSS Inc.). All data are presented as mean SD.
Differences between control and CsA-administered groups with
respect to radiotracer uptake in rats (Fig. 6) were analyzed using
an unpaired Student’s t-test with adjustments by the Bonferroni
method for multiple comparisons. The differences between non-
perfusion and brain–blood-perfusion groups with respect to
radiotracer uptake (Fig. 7) were also tested with an unpaired
Student’s t-test. P <0.05 was considered statistically significant.
[
11C]-3
The tolyl group in compound 3 was intentionally designed to
introduce a radioactive [11C]methyl group, a feasible strategy
based on the use of our 11C-labeling methods consisting of a
Pd(0)-mediated rapid C-[11C]methylation reaction.24,25 The synthe-
sis of [11C]-3 was carried out using the rapid sp3–sp2 (phenyl)
coupling of [11C]methyl iodide and the corresponding stannyl com-
pound
6
under the standard C-[11C]methylation conditions
(Scheme 1). Figure 2 shows the profile of the [11C]methylation
products by semi-preparative HPLC. The quality control after
radiopharmaceutical formulation for the animal PET study showed
that the [11C]-3 compound was synthesized with a [11C]CH3I-based
decay-corrected radiochemical yield of 30–40%, total radioactivity
3. Results and discussion
3.1. Design and synthesis of ginkgolide probes with antagonist
activity against the PAF receptor
of 2.3–3.3 GBq, specific activity (SA) of 12–45 GBq/lmol, radio-
chemical purity of 95–98%, and chemical purity of 99% (Fig. 3).
On the other hand, we focused on [18F]-235 as another ginkgolide
PET probe to reflect the in vivo behavior of the natural compound
1, since the non-radiolabeled compound 2 possesses a similar
potency to compound 1 as an antagonist of the PAF receptor.36
The synthesis of [18F]-2 was performed via nucleophilic displace-
ment of the triflate group with [18F]fluoride at the C(7) position.35
To design a ginkgolide PET probe with BBB permeability, we
focused on enhancing the hydrophobicity by introducing an alkyl
substituent at the C(10) position of ginkgolide B (1), which has rel-
atively high affinity for the PAF receptor.33,34 It is believed that the
PAF receptor is involved in several events in the CNS and that it is
an important target for the neuromodulatory effects of ginkgo-
lides.7–9 A hydroxyl group change at the C(10) position of the cor-
responding methyl ether did not influence the activity of
ginkgolide B, as shown in reports studying structure–activity rela-
tionships.33,34 Thus, we prepared 10-O-p-methylbenzyl ginkgolide
B (3) and 10-O-p-bromobenzyl ginkgolide B (4) with structures
designed to expand the PET studies.
3.3. Monkey PET imaging using [18F]-2 and [11C]-3
[
18F]-2 and [11C]-3 were dissolved in a mixed solution of intra-
lipid (20% emulsion), saline, propylene glycol, ethanol, and Tween
80 (volume ratio = 41:36:3:1:1) and intravenously injected into
live monkeys. Figures 4 and 5 show PET images of the brain of a
rhesus monkey intravenously injected with [18F]-2 (radioactivity:
We tested the ability of compounds 1, 3, and 4 to displace the
binding of [3H]WEB 2086, a PAF receptor antagonist labeled with
3H, to a cloned PAF receptor using membrane fractions from hearts
and skeletal muscles of PAF receptor-transgenic mice.29 The IC50
900 MBq, SA: 50–140 GBq/
(radioactivity: 900 MBq, SA: 12–45 GBq/
l
mol, volume: 1.0 mL) and [11C]-3
mol, volume: 1.0 mL).
l
values of compounds 1, 3, and 4 were 1.89, 0.169, and 0.205 lM
SUV images taken 34–64 min after probe injection are shown with
a horizontal section view of the brain (bottom to top). We did not
observe accumulation of [18F]-2 in the brain parenchyma (Fig. 4a),
however, a slight amount of radioactivity was observed in this
same area after administration of [11C]-3 (Fig. 5a). Time–activity
curves were prepared by quantifying the PET images of the mon-
(Fig. 1). As a result, we found that compounds 3 and 4 bound
approximately 10-fold more strongly to PAF receptors than com-
pound (1). Especially in the brain, PAF is involved in many disor-
ders such as inflammation and ischemia, and therefore the
increased antagonistic activity to PAF receptor is anticipated to
be of pharmacological benefit for such diseases.
key brain after injection with
(Figs. 4c and 5c, respectively). The abundance of the [18F]-2 and
11C]-3 probes is shown as the SUV in the cerebellum, temporal
[ [
18F]-2 and 11C]-3 probes
[
y
The SUV was calculated using the following equation: SUV = r/(a/w) [g/mL],
cortex, thalamus, and striatum, which are considered as the ROIs,
which were defined on the basis of the images obtained using a
MRI.28 Figures 4b and 5b show the PET/MRI fusion images, which
indicate these brain regions.
where r is the radioactivity concentration [Bq/mL] measured by the PET scanner
within a region of interest, a is the radioactivity of injected PET probe [Bq], and w is
the weight of the animal [g]. Most of the body has density close to 1.0 and the SUV is
normally regarded as unitless.

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H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
11CH3
(n-C4H9)3Sn
[
11C]CH3I
O
O
Pd2(dba)3/4 P(o-tolyl)3
CuCl, K2CO3
O
O
O
O
HO
HO
O
O
O
O
DMF, 5 min, 65 °C
O
O
H3C
H3C
HO
HO
O
O
O
O
6
[
11C]-3
Scheme 1. Synthesis of [11C]-3 by Pd(0)-mediated rapid C-[11C]methylation.
Thus, these quantitative analyses also denoted that [18F]-2 had
almost no BBB permeability, but [11C]-3 had a slight BBB perme-
ability. A previous PET study performed on rats injected with
[
18F]-2 revealed that there was no uptake of [18F]-2 in the rat
brain.37 Therefore, our results are in accordance with previous
findings.
3.4. Cell-permeability analyses using P-glycoprotein-expressing
cellular membranes
In parallel with the PET imaging research, we performed
cell-permeability analyses of compounds 1, 2, and 3 using a
P-glycoprotein-expressing cellular membrane. As a result, we
found that compounds 1, 2, and 3 were possible substrates of P-
glycoprotein (Table 1).30,31 Based on these results, permeability
studies were performed using membranes from (a) porcine kidney
epithelial (LLC-PK1) cells as the control cells and (b) P-glycopro-
tein-expressing cells, which are LLC-PK1 cells transfected with
human multi drug resistance (MDR1) complimentary DNA (cDNA)
to express P-glycoprotein on the apical surface. To evaluate vecto-
rial transcellular transport, each test compound (1, 2, and 3) was
added to the apical side of the membrane to then monitor their
transport across the monolayer membrane over different periods
(1, 2, and 4 h). In the same manner, the transport of each com-
pound from the basolateral side to the apical side was monitored
at the same periods. Here, we discuss the permeability using two
parameters of the apparent permeability coefficient (Papp [cm/s]),
the linear velocity and the efflux ratio.à Papp represents the linear
velocity of the permeation of each compound across the cells, while
the efflux ratio is the mean of the apical to basolateral data
(Papp[apical–basal]) and basolateral to apical data (Papp[basal–apical]),
shown as (Papp[basal–apical])/(Papp[apical–basal]).à
Figure 2. Profile of
coupled with an UV- and radio-detector.
[
11C]methylation products using
a
semi-preparative HPLC
Radiation Intensity
(a)
[11C]-3
time (min)
Typically, an efflux ratio value <1.5 is categorized as passive dif-
fusion, whereas efflux ratios that significantly exceed unity suggest
that the test compound is a substrate of the efflux transporter on
the apical surface of the membrane.30,31 As shown in the column
of (b) P-glycoprotein-expressing cells in Table 1, compound 1, with
an efflux ratio of 1.4–2.9, was considered a very weak substrate of
the P-glycoprotein, whereas the efflux ratios of compound 2 and 3
were 1.2–5.0 and 4.9–7.7, respectively, suggesting that they are
possible substrates of the P-glycoprotein. Notably, compound 3
was considered a moderate substrate for the P-glycoprotein (efflux
ratio >4.9; (b) P-glycoprotein-expressing cells in Table 1); it also
possessed a relatively high cellular permeability (Papp[apical–basal]
>6.0; (a) control cells in Table 1). However, compared to digoxin,
a representative substrate for the P-glycoprotein with an efflux
ratio >11.3 ((b) P-glycoprotein-expressing cells in Table 1),
compound 3 was considered a weaker substrate.
UV absorbance
(b)
[11C]-3
time (min)
à
The apparent permeability coefficients (Papp) were calculated using the following
Figure 3. Purity analysis of the [11C]-3 injectable solution. (a) Radio-HPLC analysis
(b) UV-HPLC analysis; the peak at 0.5–1.0 min is attributed to the mixed solution
(intralipid, saline, propylene glycol, ethanol, and Tween 80) used for preparation of
the injectable solution.
equation: Papp = dQ/dt ꢀ 1/(A ꢀ C0) [cm/s]. Where dQ/dt (lmol/s) is the permeation
rate of the compound across the cell, C0 (l
mol/cm3) is the initial concentration of the
donor compartment, and A (cm2) is the surface area of the cell monolayer. For details,
see Refs. 30,31.

H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
5153
(a) PET SUV images of the living monkey brain at 34-64 min after injection
SUV
1.2
0
(b) PET/MRI fusion images
striatum
striatum
thalamus
cerebellum
thalamus
temporal cortex
red line: brain boundary
(c) Time-activity curves of the cerebellum, temporal cortex, thalamus, and striatum
Figure 4. Monkey PET images, PET/MRI fusion images, and time-activity curves of [18F]-2.
3.5. BBB permeability in the rat brain using CsA
Based on the cell-permeability results, we further explored the
use of CsA as a P-glycoprotein inhibitor to improve BBB permeabil-
ity. CsA was administered to the rats 30 min prior to the adminis-
tration of each PET probe (properties of the injection solution per
rat, [18F]-2: radioactivity: 5 MBq, SA: 50–140 GBq/
l
mol, volume:
0.5 mL; [11C]-3: radioactivity: 20 MBq, SA: 12–45 GBq/
lmol, vol-
Results show that there were almost no changes in the abun-
dance of [18F]-2 in the brain as it remained at low level, even when
CsA was preadministered (Fig. 6a). This result could be due to the
low cellular permeability of compound 2 (Papp[apical–basal]: 0.1–0.3),
which was observed in the transcellular transport experiments
((a) control cells in Table 1). Conversely, the abundance of [11C]-3
in the brain increased dose-dependently with preadministration
of CsA (Fig. 6b). Regarding the preadministration of 40 mg/kg CsA,
the accumulation of [11C]-3 was five times higher than in the con-
trol rat. These results were similar to the trend seen for [11C]-7
(Fig. 6c). When using [11C]-7 as the reference standard, the SUV
for [11C]-3 was calculated as approximately 30% of [11C]-7 after
ume: 0.5 mL). At 5, 15, 30, and 60 min after the administration of
each PET probe, rats were decapitated to then remove the blood,
brain, and muscle, followed by measurements of the remaining
radioactivity in each of these tissues. The graphs in Figure 6 show
the tissue SUV of [18F]-2 and [11C]-3, compared to a CsA-unadmin-
istered control group, or groups where the CsA dosage was altered
(10, 25, and 40 mg/kg). Here, [11C]verapamil ([11C]-7) (properties
of the injection solution per rat, radioactivity: 20 MBq, SA: 37–
55 GBq/lmol, volume: 0.5 mL) was used as a standard substrate
to perform the brain uptake comparison study.38,39 Verapamil is
a P-glycoprotein substrate, and it is known that its uptake in the
brain increases with preadministration of CsA.40,41

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H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
(a) PET SUV images of the living money brain at 34-64 min after injection
SUV
1.2
0
(b) PET/MRI fusion images
(c) Time-activity curves of the cerebellum, temporal cortex, thalamus, and striatum
Figure 5. Monkey PET images, PET/MRI fusion images, and time–activity curves of [11C]-3.
preadministration of CsA (10, 25, and 40 mg/kg). Thus, it was deter-
mined that [11C]-3 can be positively transferred into the brain using
CsA as a P-glycoprotein inhibitor, albeit with a lower permeability
than [11C]-7. The concentration increase seen for each probe
([18F]-2, [11C]-3, and [11C]-7) in plasma after administration of
CsA could be explained by a decrease in the excretion function of
the liver and kidney due to inhibition of P-glycoprotein
activity.40,42,43
bar), there were no statistical differences between the radioactivity
of [18F]-2 in the brains of the control and the CsA-preadministered
rats (Fig. 7a). Notably, there was low radioactivity in the perfused
brains (shaded bar) of both groups (Fig. 7a). These results suggest
that [18F]-2 is not present in the brain but in the blood, and that
the BBB permeability of [18F]-2 was exceedingly low. On the other
hand, there were marked differences in the radioactivity of [11C]-3
in the brain of control and CsA-preadministered rats (non-perfu-
sion group) (Fig. 7b). More importantly, as shown in Figure 7b,
the remaining radioactivity in the rat brain after blood perfusion
(shaded bar) was almost the same than the non-perfusion group
(white bar). These results demonstrate that [11C]-3 is present in
the brain, thus confirming its BBB permeability.
In terms of the enhancement of the BBB permeability, these
results encourage the future use of other safe P-glycoprotein inhi-
bitors such as natural compounds and food constituents,44 instead
of CsA. Presumably, the flavonoid present in G. biloba extracts
could act as a weak, but effective, P-glycoprotein inhibitor that
supports the BBB permeability of ginkgolide. This is based on the
relatively weak efficacy of natural G. biloba extracts that emerges
3.6. Confirmation of radioactivity accumulation in the rat brain
after blood-perfusion treatment
To confirm the accumulation of [18F]-2 and [11C]-3 in the brain
parenchyma, we examined the remaining radioactivity of each
non-perfused and perfused rat brain, with or without preadminis-
tration of CsA (Fig. 7). At 30 min after injection of [18F]-2 or [11C]-3,
a group of rats underwent blood-perfusion with a 10% formalin
neutral buffer solution and saline through the heart vascular sys-
tem. Then, rats were decapitated and the remaining radioactivity
in the brain was measured. In the non-perfusion group (white

H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
5155
Table 1
Transcellular transport of compounds 1, 2, and 3 across (a) the control cellular membrane and (b) the P-glycoprotein-expressing cellular membranea
Compound
Concentration (
lmol/L)
Incubation time (h)
(a) Control cells
(b) P-glycoprotein-expressing cells
Papp (ꢀ10–6 cm/s)
Efflux ratio
Papp (ꢀ10–6 cm/s)
Efflux ratio
Apical to basal
Basal to apical
Apical to basal
Basal to apical
1
1
1
1
10
10
10
1
2
4
1
2
4
0.3 0.1b
0.3 0.0b
0.3 0.1
0.3 0.1
0.4 0.0
0.4 0.0
0.3 0.0
0.4 0.0
0.5 0.1
0.3 0.1
0.4 0.1
0.4 0.1
1.0
1.3
1.7
1.0
1.0
1.0
0.5 0.1b
0.7 0.1
0.7 0.0
0.4 0.1
0.6 0.0
0.7 0.0
0.7 0.1
1.4 0.7
1.7 0.5
0.8 0.1
1.5 0.3
2.0 0.7
1.4
2.0
2.4
2.0
2.5
2.9
2
1
1
1
10
10
10
1
2
4
1
2
4
0.1 0.1b
0.3 0.2b
0.3 0.1
0.3 0.1
0.3 0.0
0.3 0.1
0.2 0.1
0.3 0.0
0.5 0.1
0.3 0.1
0.3 0.1
0.3 0.1
2.0
1.0
1.7
1.0
1.0
1.0
0.2 0.2b
0.7 0.2
0.5 0.2
0.5c
1.0c
5.0
2.9
5.0
1.2
2.8
3.4
2.0c
2.5c
0.6 0.1
1.4 0.6
1.7 0.9
0.5c
0.5c
3
1
1
1
10
10
10
1
2
4
1
2
4
7.9 1.4
6.5 0.5
6.0 1.1
11.4 0.9
9.4 1.1
8.7 1.8
7.4 0.1
7.4 0.2
6.8 0.3
11.5 1.3
11.1 1.0
11.0 1.0
0.9
1.1
1.1
1.0
1.2
1.3
2.4 0.4
2.3 0.2
2.2 0.1
3.7 0.5
4.4 1.0
4.4 1.2
17.0 2.4
17.7 1.3
15.9 1.0
24.1 1.8
23.5 2.1
21.6 1.1
7.1
7.7
7.2
6.5
5.3
4.9
[3H]digoxin
1
1
1
1
2
4
1.6 0.1
1.7 0.3
1.6 0.2
1.7 0.2
2.1 0.2
2.6 0.3
1.1
1.2
1.6
1.1 0.1
1.0 0.1
0.9 0.1
12.4 0.4
12.4 0.2
12.3 0.3
11.3
12.4
13.7
a
b
c
Data are expressed as the mean SD of triplicate samples.
Lower limit of quantitation.
Mean values of duplicate samples.
(a) SUV of [18F]-2 in the blood, plasma, muscle, and brain
Control
1.2
* *
*
CsA 10mg/kg
CsA 25mg/kg
CsA 40mg/kg
*
*
*
*
0.8
0.4
0.0
*
*
*
*
*
whole blood
plasma
muscle
brain
(b) SUV of [11C]-3 in the blood, plasma, muscle, and brain
1.2
0.8
*
*
*
*
*
*
0.4
0.0
whole blood
plasma
muscle
brain
(c) SUV of [11C]-7 in the blood, plasma, muscle, and brain
2.5
2.0
1.5
1.0
0.5
0.0
*
*
whole blood
plasma
muscle
brain
Figure 6. Changes in the BBB permeability of [18F]-2, [11C]-3, and [11C]-7 in the rat brain after CsA preadministration (in these experiments, SUVs are the averaged results
from 4 rats and are presented using standard deviations). Asterisks represent values that are statistically different from the corresponding control tissues (P <0.05).

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H. Doi et al. / Bioorg. Med. Chem. 24 (2016) 5148–5157
Figure 7. Remaining radioactivity of [18F]-2 or [11C]-3 in rat brains with non-perfusion (white bar) or brain–blood-perfusion treatment (shaded bar); comparison of control
group with the CsA-preadministered group. The brain–blood-perfusion was performed with 10% formalin buffer and saline, 30 min after the injection of [18F]-2 or [11C]-3.
Asterisks represent values that are statistically differences from the corresponding control condition of non-perfusion treatment (P <0.05).
in the brain after long-term oral administration. Further studies
regarding [11C]-3 are currently in progress to examine its potential
as a drug candidate.
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