Highly efficient one-pot labeling of new phosphonium cations with fluorine-18 as potential PET agents for myocardial perfusion imaging

Article abstract of DOI:10.1021/mp500216g

Lipophilic cations such as phosphonium salts can accumulate in mitochondria of heart in response to the negative inner-transmembrane potentials. Two phosphonium salts [¹⁸F]FMBTP and [¹⁸F]mFMBTP were prepared and evaluated as potential myocardial perfusion imaging (MPI) agents in this study. The cations were radiolabeled via a simplified one-pot method starting from [¹⁸F]fluoride and followed by physicochemical property tests, in vitro cellular uptake assay, ex vivo mouse biodistribution, and in vivo rat microPET imaging. The total radiosynthesis time was less than 60 min including HPLC purification. The [¹⁸F] labeled compounds were obtained in high radiolabeling yield (～50%) and good radiochemical purity (>99%). Both compounds were electropositive, and their log P values at pH 7.4 were 1.16 ± 0.003 (n = 3) and 1.05 ± 0.01 (n = 3), respectively. Both [¹⁸F]FMBTP and [¹⁸F]mFMBTP had high heart uptake (25.24 ± 2.97% ID/g and 31.02 ± 0.33% ID/g at 5 min postinjection (p.i.)) in mice with good retention (28.99 ± 3.54% ID/g and 26.82 ± 3.46% ID/g at 120 min p.i.). From the PET images in rats, the cations exhibited high myocardium uptake and fast clearance from liver and small intestine to give high-contrast images across all time points. These phosphonium cations were radiosynthesized via a highly efficient one-pot procedure for potential MPI offering high heart accumulation and rapid nontarget clearance.

Full text of DOI:10.1021/mp500216g

Article
pubs.acs.org/molecularpharmaceutics
Highly Efficient One-Pot Labeling of New Phosphonium Cations with
Fluorine-18 as Potential PET Agents for Myocardial Perfusion
Imaging
Zuoquan Zhao,†,‡ Qian Yu,† Tiantian Mou,† Chang Liu,†,§ Wenjiang Yang,∥ Wei Fang,⊥ Cheng Peng,#
Jie Lu,† Yu Liu,∥ and Xianzhong Zhang*,§
†Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, 19 Xinjiekou
Outer Street, Beijing 100875, China
§Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular
Diagnostics, School of Public Health, Xiamen University, Xiang’an South Road, Xiamen 361102, China
⊥Department of Nuclear Medicine, Cardiovascular Institute and FuWai Hospital, Chinese Academy of Medical Sciences, Beijing
100037, China
∥Key Laboratory of Nuclear Radiation and Nuclear Energy Technology, Institute of High Energy Physics, Chinese Academy of
Sciences, Beijing 100049, China
#PET Center of Xuanwu Hospital, Capital Medical University, Beijing 100053, China
S
* Supporting Information
ABSTRACT: Lipophilic cations such as phosphonium salts
can accumulate in mitochondria of heart in response to the
negative inner-transmembrane potentials. Two phosphonium
salts [18F]FMBTP and [18F]mFMBTP were prepared and
evaluated as potential myocardial perfusion imaging (MPI)
agents in this study. The cations were radiolabeled via a
simplified one-pot method starting from [18F]fluoride and
followed by physicochemical property tests, in vitro cellular
uptake assay, ex vivo mouse biodistribution, and in vivo rat
microPET imaging. The total radiosynthesis time was less than
60 min including HPLC purification. The [18F] labeled
compounds were obtained in high radiolabeling yield (∼50%) and good radiochemical purity (>99%). Both compounds
were electropositive, and their log P values at pH 7.4 were 1.16 0.003 (n = 3) and 1.05 0.01 (n = 3), respectively. Both
[18F]FMBTP and [18F]mFMBTP had high heart uptake (25.24 2.97% ID/g and 31.02 0.33% ID/g at 5 min postinjection
(p.i.)) in mice with good retention (28.99 3.54% ID/g and 26.82 3.46% ID/g at 120 min p.i.). From the PET images in rats,
the cations exhibited high myocardium uptake and fast clearance from liver and small intestine to give high-contrast images
across all time points. These phosphonium cations were radiosynthesized via a highly efficient one-pot procedure for potential
MPI offering high heart accumulation and rapid nontarget clearance.
KEYWORDS: 18F-labeled phosphonium cation, one-pot radiosynthesis, positron emission tomography, biodistribution,
myocardial perfusion imaging agent
the high uptake in organs near the heart may compromise the
delineation of small infarcts and the diagnostic accuracy.4,5
Positron emission tomography (PET) has several technical
advantages compared with SPECT.6,7 But the half-life of
currently used PET tracers for MPI is too short, for example
[13N]ammonia (t1/2 = 10 min), [82Rb]rubidium (t1/2 = 75 s),
INTRODUCTION
■
MPI is the most important noninvasive method in the diagnosis
and treatment of coronary heart disease (CAD).1 Desired agent
for MPI should have the following properties: (1) rapid
clearance from blood; (2) quick myocardial uptake and high
myocardial uptake rate; (3) satisfactory heart-to-blood, heart-
to-liver, and heart-to-lung ratios. Currently, single-photon
emission computed tomography (SPECT) imaging with
Special Issue: Positron Emission Tomography: State of the Art
[
99mTc]sestamibi has been used as the gold standard of MPI
Received: March 23, 2014
Revised: May 9, 2014
Accepted: May 22, 2014
Published: May 22, 2014
in nuclear medicine for decades.1−4 However, the technical
limitations of SPECT imaging, such as low spatial resolution,
lack of effective methods of tissue attenuation correction, and
© 2014 American Chemical Society
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Scheme 1. 18F/11C-Labeled Analogues of Mitochondrial Complex I Inhibitor As Potential MPI Agents
Scheme 2. 18F-Labeled Lipophilic Phosphonium Cations As Potential MPI Agents
and [15O]water (t1/2 = 2 min), which limits their widespread
clinical use.8−10 [18F]Fluoride has an appropriate physical half-
life (t1/2 = 110 min), good biocompatibility, and an atomic
radius similar to that of the hydrogen atom.2,11 Therefore, the
development of novel 18F-labeled myocardial perfusion imaging
agents has become a research of interest in recent years. In the
previous studies, we and other research groups reported several
18F/11C-labeled analogues of mitochondrial complex I (MC-I)
inhibitors (Scheme 1) as potential MPI agents.2,3,12,13 Most of
those compounds have much better image quality and
relationship to myocardial blood flow than that of [99mTc]-
sestamibi.14,15
clearance23 limited their further study and use in clinic.
Therefore, it is meaningful to develop a more effective labeling
method for preparing 18F-labeled phosphonium cations with
favorable liver clearance.
In this article, two novel phosphonium cations, [18F]4-
(fluoromethyl)benzyltriphenyl phosphonium ([18F]FMBTP)
and [18F](3-(fluoromethyl)benzyl)trisphenyl phosphonium
([18F]mFMBTP), were synthesized by using a simple one-pot
procedure with high labeling yield in a short synthesis time. In
vitro cellular uptake assay, ex vivo biodistribution study in mice,
and in vivo microPET imaging in rats were carried out to
evaluate their biological properties as potential MPI agents.
Other kinds of 18F-labeled compounds of lipophilic cations
(Scheme 2), which can target mitochondria via a membrane
potential dependent mechanism in cardiomyocytes,16−19 were
also developed as potential MPI agents.1,20−23 They are
accumulated to the heart via a mechanism similar to that of
the technetium complexes ([99mTc]sestamibi and [99mTc]-
tetrofosmin) and exhibit high myocardium uptake. Unfortu-
nately, the time-consuming multiple-step radiolabeling proce-
dure,1 low radiolabeling yield,1,20,21 and unfavorable hepatic
EXPERIMENTAL SECTION
■
Materials. [18F]Fluoride was obtained from PET Center of
Xuanwu Hospital (Beijing, China). Other reagents and solvents
were purchased from commercial suppliers. Paper electro-
phoresis experiments were carried out using 0.025 mol/L
phosphate buffer (pH 7.4) and Xinhua 1# filter paper at 150 V
for 180 min. Reversed-phase high-pressure liquid chromatog-
raphy (RP-HPLC) was performed on an SHIMADZU system
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with LC-20AT pump and B-FC-3200 BIOSCAN flow-counter.
The C-18 reverse-phase semipreparative HPLC column (10 ×
250 mm, 5 μm particle size, Venusil MP-C18, Agela
Technologies Inc., USA) was eluted at a flow rate of 5 mL/
min. Labgen 7 homogenizer was purchased from Cole-Parmer
110 °C, the extra anhydrous acetonitrile (0.5 mL) was added
three times, to remove the residual water. After that, 1,4-
bis(bromomethyl)benzene (for [18F]BMFMB) or 1,3-bis-
(bromomethyl)benzene (for [18F]mBMFMB) was dissolved
in anhydrous acetonitrile (3 mg in 0.5 mL) and added to the
residuals. After vibrating at 100 °C for 6 min, the reaction
mixture was cooled to room temperature. The crude product
could be used for next step without further purification. The
radiochemical yields were calculated by analytical HPLC. The
column was eluted with water (solvent A) and acetonitrile
(solvent B) at a flow rate of 1.0 mL/min (gradient method).
Gradient: 0−3 min, 95−60% A; 3.01−7 min, 60−30% A; 7.01−
20 min, 0% A.
1
Instrument Co (USA). H NMR spectra were recorded on a
Bruker (400 MHz) spectrometer. Mass spectra were recorded
using a Bruker Compass Data analysis 4.0. Chemical shifts are
reported in δ (ppm) values.
Kunming mice (18−20 g) and Spraque-Dawley rats (250−
260 g) were obtained from the Animal Center of Peking
University. Beagle dogs were obtained from the Beijing Rixin
Technology Co., Ltd. All biodistribution studies were
performed under a protocol approved by Beijing Admin-
istration Office of Laboratory Animal (BAOLA).
Chemistry: Synthesis of Nonradioactive Reference
Compounds. 4-(Fluoromethyl)benzyltriphenyl phosphonium
([19F]FMBTP) and 3-(fluoromethyl)benzyltriphenyl phospho-
nium ([19F]mFMBTP) were synthesized as reference com-
pounds with similar two-step procedure.
Preparation of [18F]FMBTP and [18F]mFMBTP. Triphenyl-
phosphine (5 mg in 0.5 mL of anhydrous acetonitrile) was
added to the unpurified crude ([18F]BMFMB or [18F]-
mBMFMB) and stirred at 90 °C for 15 min. After being
cooled to room temperature, the final reactant was injected
onto a semipreparative HPLC column for purification. The
column was eluted with PBS (pH = 7, 0.02 mol/L) and
acetonitrile (v/v = 1:1) at a flow rate of 3.0 mL/min (isocratic
purification method). The appropriate fraction was collected
and evaporated to remove acetonitrile, and then the activity was
reconstituted in phosphate-buffered saline and passed through a
0.22 μm Millipore filter into a sterile vial for in vivo applications.
The radiochemical purity was assayed by analytical HPLC by
using PBS (pH = 7, 0.02 mol/L) and acetonitrile (v/v = 1:1) at
a flow rate of 1.0 mL/min (isocratic analysis method).
Physicochemical Property Studies. The octanol/water
partition coefficient was measured following 5−8 min of
vigorous vortex mixing of 600 μL of n-octanol and 600 μL of
phosphate buffer (0.025 mol/L, pH 7.4) with [18F]FMBTP or
[18F]mFMBTP in a tube. The tube was centrifuged at 14000
rpm for 5 min, and the counts in 500 μL aliquots of both
organic and aqueous layers were determined. Then the organic
and aqueous phases were mixed again, and the above operation
was repeated three times to ensure that the ratio of counts in
organic and aqueous phases remained unchanged. The partition
coefficient (P) was calculated using the following equation: P =
(cpm in the organic phase − background cpm)/(cpm in the
aqueous phase − background cpm). The partition coefficient
value was expressed as log P.
tert-Butylammonium fluoride (TBAF, 6 mmol) and acetoni-
trile (anhydrous, 5 mL) were placed into a three-necked flask
(100 mL), and the liquid was evaporated at 110 °C under a
stream of nitrogen. Two azeotropic evaporations with
acetonitrile (anhydrous, 2 × 5 mL) were carried out at 110
°C under a stream of nitrogen, and 1,4-bis(bromomethyl)-
benzene (1.32 g, 5 mmol) in acetonitrile (anhydrous, 30 mL)
was added at 60 °C. The mixture was kept at 100 °C overnight
and concentrated. [19F]BMFMB was obtained as a colorless oil
(0.4 g, 40%) after silica gel chromatography (mineral ether).
[19F]mBMFMB was obtained as a colorless oil (0.18 g, 45%) by
using a procedure similar to that described above, except
starting from 1,3-bis(bromomethyl)benzene (0.528 g, 2 mmol).
1
[19F]BMFMB and [19F]mBMFMB were characterized by H
NMR and 19F NMR (see Supporting Information).
[19F]BMFMB or [19F]mBMFMB (0.14 g, 0.7 mmol),
triphenylphosphine (0.24 g, 0.9 mmol), and anhydrous
acetonitrile (25 mL) were placed into a three-necked flask
(100 mL) stirred at 85 °C for 5 h. After cooled to room
temperature, the reactant was concentrated under reduced
pressure. After that, [19F]FMBTP was obtained as a white solid
(0.11 g, 35%) after eluted with the mixture of ethyl acetate and
petroleum ether =1:1 (v/v). While for [19F]mFMBTP, it was
obtained as a white solid (0.1 g, 32%) after silica gel
For paper electrophoresis experiments, the radiotracer
([18F]FMBTP or [18F]mFMBTP) was spotted on a chroma-
tography paper strip (10 cm × 1 cm), which was pretreated
with phosphate buffer (0.025 mol/L, pH = 7.4). The strip was
developed in phosphate buffer (0.025 mol/L, pH = 7.4) at 150
V for 2 h. Then the radioactivity distribution of radiotracer on
the strip was determined.
1
chromatography (CH2Cl2 /CH3OH = 20:1 (v/v)). H NMR
1
and 19F NMR for [19F]FMBTP: H NMR (400 MHz, CDCl3)
δ: 5.22 (s,1H), 5.34 (s,1H)), 5.46 (d,2H), 7.12−7.17 (m, 4H),
7.16−7.65 (m, 15H); 19F NMR (400 MHz, CDCl3) δ:
−208.35; HRMS: m/z[M+] calculated for [19F]FMBTP:
385.1516, found 385.1175. 1H NMR and 19F NMR for
[19F]mFMBTP: 1H NMR (400 MHz, CDCl3) δ: 5.09
(s,1H)), 5.21 (s,1H)), 5.51 (d,2H), 7.17−7.24 (m, 4H),
7.62−7.64 (m, 15H); 19F NMR (400 MHz, CDCl3) δ:
−208.27; HRMS: m/z[M+] calculated for [19F]mFMBTP:
385.1516, found 385.1511.
For in vitro stability study, radiotracer ([18F]FMBTP or
[18F]mFMBTP) was incubated in 1 mL of water at room
temperature or in 0.5 mL of murine plasma at 37 °C for 3 h,
respectively. Plasma proteins were precipitated by adding 150
μL of acetonitrile and removed by centrifugation. The
radiochemical purity was assayed by HPLC using the isocratic
analysis method.
Radiochemistry. Preparation of [18F]BMFMB and [18F]-
mBMFMB. The labeling procedure of [18F]BMFMB (or
[18F]mBMFMB) is similar to that of published previously.24,25
While in our study, the amount of reagents were modified to
obtain high radiochemical yields (see Supporting Information).
Briefly, after the solvent of [18F]fluoride eluate (1.5 mg of
K2CO3 in 0.3 mL of H2O and 2 mg of Kryptofix 2.2.2 in 1 mL
of acetonitrile) was evaporated under a stream of nitrogen at
Cell Uptake Studies. The cell uptakes of lipophilic cations
of [18F]FMBTP and [18F]mFMBTP were determined in rat
embryonic cardiomyoblast cell line (H9c2) and mouse normal
fibroblast cell line (NIH/3T3), respectively. All cells were
grown in Dulbecco’s modified Eagle medium high glucose
(containing KCl, 5.3 mmol/L, and NaCl, 110.34 mmol/L), plus
10% fetal bovine serum and 1% penicillin−streptomycin at 37
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°C in 5% CO2 and 95% air. Twenty-four hours before the
experiment, the H9c2 and NIH/3T3 cells were seeded in 48
well plates (0.05 M cell/well, 0.5 mL) and incubated at 37 °C
to form confluent monolayers. After the radiotracer
([18F]FMBTP or [18F]mFMBTP, 18.5 kBq/100 μL) was
added, cells were incubated at 37 °C for 1 h. Then the
radioactive medium was removed, and the wells were washed
two times with cold phosphate-buffered saline (0.01 mol/L;
pH, 7.4; 0.2% BSA). The cells were lysed by treated with 0.5
mL of 1 mol/L NaOH for 5 min at room temperature and
transferred to tubes, and the radioactivity was measured using a
gamma counter. The cell uptakes of [99mTc]sestamibi (11.1
kBq/100 μL) in H9c2 and NIH/3T3 cells were also performed
for comparison.
The mitochondrial membrane potential (MMP) dependent
cellular uptake of radiotracers was further evaluated with H9c2
cells pretreated with carbonyl cyanide m-chlorophenylhydra-
zone (CCCP), which is a protonophore that selectively
abolishes the mitochondrial membrane potential.26 The
radiotracer was dissolved in dimethyl sulfoxide and diluted to
the desired concentration with low-K+ HEPES buffer (NaCl,
135 mmol; KCl, 5 mmol/L; CaCl2, 1.8 mmol/L; MgSO4, 0.8
mmol/L; HEPES, 50 mmol/L; dextrose, 5.5 mmol/L; pH,
7.4).27 The final concentration of dimethyl sulfoxide was
<0.1%. CCCP solution (1 μM) was added to 5.0 × 104 cells for
30 min at 37 °C before the start of the experiment. After
incubation with [18F]FMBTP ([18F]mFMBTP or [99mTc]-
sestamibi), the cells were washed and lysed to count the
radioactivity by a gamma counter.
About 55 MBq of [18F]mFMBTP in 2 mL of saline solution
was injected. Whole-body imaging was performed at 10, 30, 60,
and 120 min after injection. PET data were acquired on a PET/
CT system (Biograph 64, Siemens). Whole-body scanning
involved 4 bed positions, with each scanned for 2 min. Regions
of interest were drawn on the myocardium, liver, and lung.
Standardized uptake value was calculated as [mean region-of-
interest count (cps/pixel) × body weight (kg)]/[injected dose
(mCi) × calibration factor (cps/pixel)]. The myocardium/liver
and myocardium/lung standardized uptake value ratios at each
time point were evaluated.
RESULTS
■
Chemistry. The intermediate products of [19F]BMFMB
and [19F]mBMFMB were synthesized and characterized by H
1
NMR and 19F NMR (see Supporting Information). Non-
radioactive references [19F]FMBTP and [19F]mFMBTP were
characterized by 1H NMR, 19F NMR and HRMS. The synthetic
routes are shown in Figure 1. The chemical yields of
Ex Vivo Biodistribution Studies. About 185 kBq
[18F]FMBTP or [18F]mFMBTP (0.1 mL) was injected through
the tail vein of wild type Kunming mice (n = 5). The mice were
sacrificed at 5, 30, 60, and 120 min after injection. The tissues
and organs of interest were collected, wet weighed, and counted
in gamma counter (WIZARD1470, PerkinElmer, USA). The
percentage of injected dose per gram (% ID/g) for each sample
was calculated by comparing its activity with appropriate
standard of injected dose (ID), the values expressed as mean
standard deviation (SD).
MicroPET Imaging in Rat. All the animal protocols were
approved by the Institute’s Animal Care and Use Committee.
Normal male Spraque-Dawley rat (250−260 g) was anes-
thetized with isoflurane and placed near the center of the field
of view of the PET scanner (Siemens Inveon). About 21 MBq
of [18F]mFMBTP was injected intravenously. Static PET scans
were performed to evaluate myocardial uptake at 5, 30, 60, and
120 min after the tracer injection, and the each scan time was
10 min, respectively. Dynamic small-animal PET images were
acquired for 120 min (6 s × 10 frames, 60 s × 4 frames, 300 s ×
6 frames, 600 s × 9 frames) after injection. The images were
reconstructed using a two-dimensional ordered-subsets expect-
ation maximization (2-D OSEM) algorithm without attenu-
ation or scattering correction. Three-dimensional regions of
interests (3D-ROIs) were placed on for the ROI drawing.
PET/CT Imaging in Dog. Animal procedures were
performed following National Institutes of Health guidelines
and were approved by FuWai Hospital, Chinese Academy of
Medical Science.
Figure 1. Synthetic routes of [18/19F]FMBTP and [18/19F]mFMBTP.
[19F]BMFMB, [19F]mBMFMB, [19F]FMBTP, and [19F]-
mFMBTP were 40%, 45%, 35%, and 32%, respectively. The
chemical purities of nonradioactive references [19F]FMBTP
and [19F]mFMBTP were calculated as >98% from the HPLC
chromatogram (λ = 254 nm) (see Supporting Information:
Figure S1 and S2). Besides that, there is more than 2 min
difference between the retention time of intermediates and final
compound, meaning that the intermediates could be separated
clearly from the final reference compounds.
Radiochemistry: Preparation of Radiointermediates.
For the synthesis of [18F]BMFMB, 1,4-bis(bromomethyl)-
benzene was reacted with non-carrier-added [18F]fluoride in the
presence of Kryptofix 2.2.2 and K2CO3 (Figure 1). HPLC
analysis revealed that the [18F]BMFMB exhibited identical
retention time with a fully characterized [19F]BMFMB (see
Supporting Information: Figure S1), which confirmed that the
radiointermediate product was synthesized successfully. The
high radiochemical yield (85%) was obtained within 15 min
synthesis time from the addition of [18F]fluoride.
For the synthesis of [18F]mBMFMB, even higher radio-
chemical yield (92%) was also obtained with a procedure
similar to that of [18F]BMFMB, instead of using 1,3-
bis(bromomethyl)benzene as reactant. The analysis HPLC
profiles are shown in Figure S2 (Supporting Information).
Preparation of [18F]FMBTP and [18F]mFMBTP. Based on
the convenient labeling of radiointermediates, the total
For the whole-body PET/CT study, a healthy Beagle dog
(15 kg) was anesthetized by a mixture of ketamine (25 mg/kg)
and diazepam (1.1 mg/kg). Anesthesia was supplemented as
needed. The animal was placed prone on the PET/CT bed, and
a venous catheter was established for radiotracer injection.
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Figure 2. HPLC chromatograms of compounds [18F]FMBTP (A) and [18F]mFMBTP (D) and the profiles of stability study of [18F]FMBTP and
[18F]mFMBTP after storage in water at room temperature for 3 h (B and E), and after incubation in murine plasma at 37 °C for 3 h (C and F)
respectively. All the radiotracers were analyzed by using the isocratic HPLC method.
synthesis times of [18F]FMBTP and [18F]mFMBTP (from
[18F]fluoride) were about 50 and 55 min including HPLC
purification, respectively. Their decay-corrected radiochemical
yields were about 52 9.3% (n = 7, for [18F]FMBTP) and 50.6
6.9% (n = 7, for [18F]mFMBTP). The retention times of
[18F]FMBTP and [18F]mFMBTP (12.30 and 12.82 min,
respectively) also agreed well with the corresponding non-
radioactive references [19F]FMBTP and [19F]mFMBTP (see
Supporting Information: Figure S1 and S2). Both [18F]FMBTP
and [18F]mFMBTP demonstrated excellent radiolabeling yields
(>50%) and radiochemical purities (>99%). The specific
activities of [18F]FMBTP and [18F]mFMBTP were estimated
by HPLC analysis to be about 30 GBq/μmol and 38 GBq/
μmol, respectively.
comparable to the [18F]FETMP (1.27
0.01)28 and (6-
[18F]fluorohexyl)triphenylphosphonium salt ([18F]3) (1.78
0.05).5 In paper electrophoresis, more than 98% [18F]FMBTP
and [18F]mFMBTP were accumulated on the negative part,
indicating that both of them were electropositive compounds.
For the in vitro stability studies, both of these radiotracers
were intact in water solution (room temperature) and murine
plasma (37 °C) after 3 h incubation (Figure 2), suggesting that
they had very good stability in vitro.
In Vitro Cell Uptake Assays. In this study, the binding
abilities of [18F]FMBTP, [18F]mFMBTP, and [99mTc]sestamibi
to the H9c2 cells (which have high mitochondrial membrane
potential) and NIH/3T3 cells were evaluated and compared.
For [18F]FMBTP, cell uptake in H9c2 cells was much higher
(>2.67-fold) than that in NIH/3T3 cells after incubation for 1
h. When the cells were pretreated with 1 μM CCCP for 0.5 h,
cell uptake of [18F]FMBTP was significantly inhibited to about
Physicochemical Property Study. The partition coef-
ficients (log P) of [18F]FMBTP and [18F]mFMBTP were
measured as 1.16 0.003 (n = 3) and 1.05 0.01 (n = 3),
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70% (P < 0.01) (Figure 3). Analogously, cell uptake of
blood ratios were 5.04, 5.80, and 32.9, respectively, at 30 min
p.i., and increased to 32.5, 7.61, and 151.9, respectively, at 120
min p.i.
[18F]mFMBTP also higher (>1.52-fold) in H9c2 cells than that
When compared with [18F]FMBTP, [18F]mFMBTP had
higher heart uptake (31.02 0.33% ID/g at 5 min p.i.) and
better target-to-nontarget ratios at earlier time points, while at
later time points, the heart uptake and target-to-nontarget ratios
have no obviously difference. Both of these new cations have
moderate muscle uptakes at all time points after injection (6.91
1.04% ID/g for [18F]FMBTP and 4.29 0.67% ID/g for
[18F]mFMBTP at 60 min p.i., respectively) (Table 1), similar to
that of previously reported lipophilic cations of [18F]FPTP
(6.16 1.78% ID/g at 60 min p.i.)10 and [18F]3 (4.53 0.54%
ID/g at 60 min p.i.),5 respectively. For all of the other tissues,
these two radiotracers showed comparable radioactivity uptake
except that [18F]mFMBTP had less bone uptake at the later
time points.
Above all, [18F]mFMBTP seems to be promising as a
potential myocardial perfusion imaging agent, this encourage us
to further evaluate it by in vivo PET imaging in rat and dog.
MicroPET Imaging Studies. MicroPET studies using
[18F]mFMBTP showed an excellent image quality in rat at 5
min, 30 min, 60 min, and 120 min after iv injection. Decay
corrected coronal images that contain the heart are shown in
Figure 4; high contrast images were obtained with sustained
high myocardium uptake and extremely low liver and lung
uptake. From the time−activity curves derived from dynamic
PET imaging (Figure 5), the highest myocardium uptake
(SUV) was reached rapidly at very early time after injection and
kept high across all time points to 120 min p.i. For liver uptake,
it peaked at 5 min p.i. and was followed by rapid clearance.
After 30 min p.i., it was even lower than that of lung uptake.
Sustained low lung uptake was observed from about 2 min p.i.
with SUV value of 0.5. The heart/liver ratio increased obviously
due to the quick clearance from liver (1.37, 6.62, 12.06, and
18.64 at 5, 30, 60, and 120 min p.i., respectively). High heart/
lung ratios (3.87, 5.39, 6.03, and 6.61 at 5, 30, 60, and 120 min
p.i., respectively) were also obtained at all time points after
injection. This agreed with the results of ex vivo biodistribution
very well. It is worth mentioning that almost no bone uptake of
[18F]mFMBTP was observed in PET imaging, although high
bone uptake was found in the biodistribution study of mice.
PET/CT Imaging in Dog. The representative whole-body
2D projection images of [18F]mFMBTP in a healthy Beagle dog
are shown in Figure 6. The heart could clearly be seen from 10
Figure 3. Cell uptake of [18F]FMBTP, [18F]mFMBTP, and [99mTc]-
sestamibi in H9c2, H9c2 cells treated with 1 μM protonophore CCCP,
and NIH/3T3 cells at 1.0 h in a 37 °C incubator.
in NIH/3T3 cells after incubation for 1 h. And the uptake in
H9c2 cells could be inhibited to about 48% (P < 0.01) when
pretreated with 1 μM CCCP for 0.5 h (Figure 3).
As a known membrane potential dependent tracer,29
[
99mTc]sestamibi was set as positive control. It had more
than 2.7-fold higher uptake in H9c2 cells than that in NIH/3T3
cells after incubation for 1 h. And uptake of [99mTc]sestamibi in
H9c2 cells was also obviously inhibited by 1 μM CCCP (P <
0.01) (Figure 3). These results fully demonstrate that the two
novel 18F-labeled cations reported here accumulate in the
myocardial cells through the mitochondrial membrane
potential, like [99mTc]sestamibi.
Biodistribution in Normal Mice. All the animal studies
were carried out in compliance with relevant national laws
relating to the conduct of animal experimentation.
The biological distribution results in mice are shown in Table
1. [18F]FMBTP had high heart uptake (25.24 2.97% ID/g at
5 min post injection, p.i.) and very good retention (28.99
3.54% ID/g at 120 min p.i.). On the other hand, the clearance
of [18F]FMBTP from the nontarget tissues was very fast. For
example, the liver uptake was decreased dramatically from
14.93 1.46% ID/g (at 5 min p.i.) to 0.89 0.29% ID/g (at
120 min p.i.). The heart-to-liver, heart-to-lung, and heart-to-
Table 1. Biodistribution Results of [18F]FMBTP and [18F]mFMBTP in Mice at 5, 30, 60, and 120 min after Injection
(Expressed as % ID/g SD, n = 5)
5 min
[18F]mFMBTP
30 min
60 min
120 min
organ
heart
[18F]FMBTP
[18F]FMBTP
[18F]mFMBTP
[18F]FMBTP
[18F]mFMBTP
[18F]FMBTP
[18F]mFMBTP
25.24 2.97
14.93 1.46
4.84 0.87
6.53 0.43
5.35 1.32
7.12 1.42
73.62 5.4
1.82 0.14
1.69
31.02 0.33
14.94 1.39
5.75 0.64
7.92 0.34
6.98 1.14
6.59 0.3
51.11 8.25
2.14 0.16
2.08
26.08 2.9
5.16 0.64
3.93 0.83
4.49 0.8
4.95 0.53
19.45 1.93
32.28 1.63
0.79 0.06
5.04
27.39 1.46
6.29 1.35
6.09 0.96
5.36 0.68
6.53 1.65
18.13 1.27
36.22 3.29
1.15 0.14
4.84
31.17 1.5
1.89 0.32
2.83 0.51
4.1 0.69
6.91 1.04
20.16 3.9
17.1 3.72
0.41 0.01
16.43
28.30 2.36
2.39 0.56
3.83 0.53
5.08 0.32
4.29 0.67
14.2 1.13
19.73 1.69
0.61 0.05
11.84
28.99 3.54
0.89 0.29
2.37 0.55
3.81 0.97
5.81 1.59
22.61 5.5
13.24 1.31
0.19 0.02
32.5
26.82 3.46
1.02 0.2
2.41 0.22
2.68 0.46
5.13 0.3
11.69 2.74
11.04 1.46
0.32 0.05
26.25
liver
spleen
lung
muscle
bone
kidney
blood
heart/liver
heart/lung
heart/blood
3.86
3.91
5.80
5.11
7.46
5.57
7.61
9.97
13.87
14.44
32.90
23.82
74.74
46.39
151.90
83.98
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Figure 4. Coronal microPET images in normal rat. The heart was visible with excellent ratios of heart/liver and heart/lung, and fast clearance from
small intestine at 5, 30, 60, and 120 min after iv injection of [18F]mFMBTP, respectively.
DISCUSSION
■
The radiointermediates of [18F]BMFMB and [18F]mBMFMB
had been reported previously by Vries et al.24,25 In the
literature, the radiointermediates were synthesized and purified
with HPLC in approximately 55 min with low radiochemical
yield (12% for [18F]BMFMB and 26% for [18F]mBMFMB),
while in this study, the optimized labeling conditions were
obtained with less precursor (3 mg) and Kryptofix 2.2.2 (2 mg)
(see Supporting Information: Table S1). The radiochemical
yields were increased significantly to 85% ([18F]BMFMB) or
92% ([18F]mBMFMB) by HPLC analysis with decay-
correction, and the synthesis time was also shortened obviously
to about 15 min (from [18F]fluoride). Over all, the advantages
of this highly efficient one-pot labeling method include smaller
amounts of Kryptofix 2.2.2 and precursor, shorter synthesis
time, and much higher radiochemical yield than that of previous
study.
Figure 5. Time−activity curves generated from dynamic PET images.
[18F]mFMBTP accumulated specifically in the heart. The [18F]-
mFMBTP had excellent heart/liver and heart/lung ratios and in liver
and lung was washed out rapidly but was retained in the myocardium
for the whole time.
When compared with the previously reported phosphonium
cations, [18F]FMBTP and [18F]mFMBTP have signicant
advantages in the aspects of synthesis time and labeling yield
due to the highly efficient one-pot labeling of radio-
intermediate. For [18F]FTPP, the synthesis time was reported
as 120 min with 10% labeling yield.23 For [18F]FBnTP, it was
synthesized in 82 min with only 6% radiochemical yield.
Recently, Kim et al.5 reported a new cation, (6-[18F]-
fluorohexyl)triphenylphosphonium salt ([18F]3), which was
prepared in a short time of 56 min while the radiochemical
yield still needs to be improved (15−20%).
In this study, the preliminary properties and feasibilities of
[18F]FMBTP and [18F]mFMBTP for myocardial perfusion
imaging were investigated in vitro and in vivo. In the ex vivo
bisdistribution study, both [18F]FMBTP and [18F]mFMBTP
had comparable target/nontarget ratios to [18F]FTPP and even
to 120 min after injection, and heart uptake had not obviously
decreased. Radioactivity accumulated strongly in the kidney and
gall bladder at all time points and gradually increased in the
bladder from 10 to 120 min. Very low bone and muscle uptakes
were observed from 30 min, and the heart-to-bone and heart-
to-muscle ratios, which were derived from PET imaging, were
10.37, 13.16, 12.47, and 12.96 and 17.00, 17.74, 19.95, and
24.20 at 10, 30, 60, and 120 min after injection, respectively.
Other organs and tissues had low background uptake because
of the excellent metabolic properties of the compound. The
heart/liver and heart/lung standardized uptake value ratios
were calculated as 1.54 and 6.00 at 10 min after injection, 2.83
and 15.19 at 30 min after injection, 3.98 and 38 at 60 min after
injection, and 7.76 and 35.28 at 120 min after injection,
respectively. The results of PET imaging in dog were coincident
with the biodistribution results in rat and mice.
Figure 6. Whole-body 2D projection PET/CT images in normal Beagle dog at 10, 30, 60, and 120 min after iv injection of [18F]mFMBTP,
respectively.
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higher at later time points.21 When compared to [18F]-
FETMP,28 faster liver clearance for [18F]FMBTP and [18F]-
mFMBTP was observed. The liver uptakes of [18F]FETMP
were 16.25 2.77% ID/g at 10 min p.i. and 10.38 1.94% ID/
g at 120 min p.i., and only about 64% liver uptake was cleared
after 2 h injection, obviously slower than that of both
[18F]FMBTP and [18F]mFMBTP (more than 90% liver
accumulation was cleared from 5 to 120 min p.i.).
The accumulation of activity in kidney may due to the
electropositivity of [18F]FMBTP and [18F]mFMBTP, because
kidney tubules have some compounds with negative charges
which prefer to attract molecules with positive charges. Without
doubt, the [18F]FMBTP and [18F]mFMBTP were excreted
through kidney and finally washed out with urine. High bone
uptakes were also found in mouse biodistribution for both
[18F]FMBTP and [18F]mFMBTP, and the activity increased
continuously with time for [18F]FMBTP. The reason may due
to the defluorination of tracers in vivo (see Supporting
Information: Figure S3). Fortunately, there was no obvious
bone uptake in the PET imaging in rat and dog.
ASSOCIATED CONTENT
* Supporting Information
NMR characterization, optimized radiolabeling conditions,
HPLC analysis, urinary metabolites in mice, and biodistribution
in rat. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*School of Public Health, Xiamen University, Xiang’an South
Rd., Xiang’an district, Xiamen 361102, China. E-mail:
zhangxzh@xmu.edu.cn. Phone: +86(592)2880645. Fax:
+86(592)2880645.
■
Notes
The authors declare no competing financial interest.
‡First author. College of Chemistry, Beijing Normal University,
19 Xinjiekou Outer St., Beijing 100875, China. E-mail:
418zhaozuoquan@163.com. Phone: +8610-58802842. Fax:
+8610-62208126.
In the study of [18F]3, Kim et al.5 claimed that highly
lipophilic structures (such as benzene rings) should not be
adopted for the radiolabeling of phosphonium salts because
they could increase liver uptake. But in this study, although
lipophilic structures were introduced as labeling intermediates
in both [18F]FMBTP and [18F]mFMBTP, their liver uptake
was cleared even much faster than that of [18F]3. For instance,
the liver uptake of [18F]FMBTP was 5.16, 1.89, and 0.89% ID/
g at 30, 60, and 120 min respectively, while that of [18F]3 was
4.52, 3.70, and 2.84% ID/g at 30, 60, and 120 min, respectively.
Furthermore, the introduction of the aromatic 18F-labeling
moiety does not increase the log P value of the tracers
([18F]FMBTP and [18F]mFMBTP have a log P similar to that
of [18F]FETMP28 and [18F]35).
ACKNOWLEDGMENTS
■
We thank Yuan Chen, Zhide Guo, Pu Zhang, Yi Sun, and Lin
Wang for their generous help with biodistribution and PET
imaging. This project was sponsored by the National Key Basic
Research Program of China (2014CB744503) and the National
Natural Science Foundation of China (21271030, 20871020,
81301251) and supported partially by Department of Nuclear
Medicine of Peking Union Medical College Hospital.
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