PET probe detecting non-small cell lung cancer susceptible to epidermal growth factor receptor tyrosine kinase inhibitor therapy

Article abstract of DOI:10.1016/j.bmc.2018.02.007

Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR-TKIs) are used as molecular targeted therapy for non-small cell lung cancer (NSCLC) patients. The therapy is applied to the patients having EGFR-primary L858R mutation, but drug toleran

Full text of DOI:10.1016/j.bmc.2018.02.007

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
PET probe detecting non-small cell lung cancer susceptible to epidermal
growth factor receptor tyrosine kinase inhibitor therapy
a
a
Akira Makino a,b, , Anna Miyazaki , Ayaka Tomoike , Hiroyuki Kimura a,c, Kenji Arimitsu c,
⇑
Masahiko Hirata d, Yoshiro Ohmomo d, Ryuichi Nishii e, Hidehiko Okazawa b, Yasushi Kiyono b,
Masahiro Ono a, Hideo Saji a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Adachi-cho, Sakyo-ku, Kyoto 6068501, Japan
b Biomedical Imaging Research Center (BIRC), University of Fukui, 23-3 Matsuoka-shimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 9101193, Japan
c Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 6078414, Japan
d Graduate School of Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, Osaka 5691041, Japan
e National Institute of Radiological Sciences, Chiba 2638555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR-TKIs) are used as molecular
targeted therapy for non-small cell lung cancer (NSCLC) patients. The therapy is applied to the patients
having EGFR-primary L858R mutation, but drug tolerance caused by EGFR-secondary mutation is
occurred within one and half years. For the non-invasive detection of the EGFR-TKIs treatment positive
patients by positron emission tomograpy (PET) imagaing, fluorine-18 labeled thienopyrimidine deriva-
tive, [18F]FTP2 was newly synthesized. EGFR inhibition assay, cell uptake study, and blocking study
indicated [18F]FTP2 binds with high and selective affinity for EGFR with L858R mutation, and not with
L858R/T790M dual mutations. On animal PET study using tumor bearing mice, H3255 cells expressing
L858R mutated EGFR was more clearly visualized than H1975 cells expressing L858R/T790M dual
mutated EGFR. [18F]FTP2 has potential for detecting NSCLC which is susceptible to EGFR-TKI treatment.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 2 November 2017
Revised 6 February 2018
Accepted 7 February 2018
Available online xxxx
Keywords:
Epidermal growth factor receptor
EGFR L858R and T790M mutations
Tyrosine kinase inhibitor
Positron emission tomography
In vivo imaging
1. Introduction
60% of the cause is a secondary mutation of EGFR-T790M.5–7 On
clinical site, genetic examination by biopsy will be conducted after
World Health Organization (WHO) reported lung cancer is the
leading cause of cancer death worldwide, accounting for 1.69 mil-
lion deaths in 2015. It can be categorized to small cell lung cancer
(SCLC) and non-small cell lung cancer (NSCLC), and the treatment
is varied depending on the cancer type and stage. In case of stage
IIIB or IV NSCLC, the epidermal growth factor receptor-tyrosine
kinase inhibitor (EGFR-TKI) such as gefitinib and erlotinib is one
treatment option. EGFR-TKIs can block signal transmission for can-
cer cells proliferation and suppress the growth.1 Especially, they
work effectively to the patient whose EGFR-tyrosine kinase have
primary mutation such as L858R in exon 21 and exon 19 dele-
tion.2–4 Therefore, detection of the EGFR mutation by biopsy test
is essential for the treatment policy determination. However, the
problem is EGFR-TKI treatment induces drug tolerance within
about one and a half years after the start of the treatment, and
cancer exacerbation is occurred during the treatment, and pres-
ence or absence of the EGFR secondary mutation is investigated.
This is because biopsy test is invasive and cannot be performed
so frequently. When the T790M mutation becomes positive, the
treatment is switched to platinum preparations and/or osimer-
tinib,8–10 which is EGFR T790M mutation-positive metastatic
NSCLC therapeutic drug. Recently, liquid biopsy is also utilized
for the detection of T790M mutation, but there is a time lag
between occurrence of the second mutation caused drug tolerance
and switching of the treatment policy.
As PET tracers detecting EGFR overexpressing on NSCLC, radio-
labeled gefitinib and erlotinib derivatives are developed.11–13 How-
ever, it has also been reported that these probes are not suitable for
detecting drug tolerance of EGFR-TKIs.14 Development of a new
PET probe which can detect EGFR-TKIs treatment response NSCLC
is important because periodic PET examination for patients under-
going EGFR-TKI treatment enables early detection of the drug tol-
erance, and helps prompt modification of the treatment strategy.
In our group, radiofluorinated 4-(anilino)pyrido[3,4-d]pyrimidine
derivative, [18F]APP-1 was synthesized which shows potential to
⇑
Corresponding authors at: Biomedical Imaging Research Center (BIRC), Univer-
sity of Fukui, 23-3 Matsuoka-shimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 9101193,
Japan (A. Makino).
E-mail addresses: amakino@u-fukui.ac.jp (A. Makino), hsaji@pharm.kyoto-u.ac.
jp (H. Saji).
https://doi.org/10.1016/j.bmc.2018.02.007
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Makino A., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.007

2
A. Makino et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
be used as a PET tracer to discriminate between L858R and L858R/
T790M mutant EGFRs in NSCLC.15 However, APP-1 has weak inhi-
bition ability against L858R/T790M mutated EGFR (IC50 = 326 64
nM). Therefore, development of a new PET tracer with higher selec-
tivity against L858R and L858R/T790M mutated EGFRs is per-
formed in this study.
EGFR were summarized in Table 1. FTP2 and FTP5 showed low
IC50 values against wild type and L858R mutated EGFRs. IC50 value
of FTP2 against L858R mutated EGFRs was 9 nM, whose value is
lower than that of gefitinib. The influence of the methylol unit sub-
stitution to methyl group is limited, but IC50 value of FTP5 against
L858R mutated EGFRs was increased to 27 nM. The high IC50 value
of FTPs3 and 4 indicate Michael acceptor group in FTPs2 and 5
works effectively for the kinase activity inhibition. Importantly,
almost all compounds didn’t show inhibitory activity against
EGFRs including T790M gefitinib negative mutation. IC50 value of
FTP2 against T790M EGFR was 8966 nM, which is ca. 1000 times
higher than that against L858R EGFR. Because of the excellent
IC50 value of FTP2 against L858R mutated EGFR, FTP2 was selected
for the further evaluations.
2. Results and discussion
2.1. Probe design
Thienopyrimidine derivatives, which are reported to be inhibi-
tors against wild type and primary mutated EGFRs, but not against
secondary mutated EGFR, was selected as the mother nucleus
structure on this study.16–19 Fluorine-18 was chosen as positron
nuclides, which has a relatively long half-life time of 110 min. Can-
didate compounds designed (FTPs1–5) were listed in Fig. 1. FTP1
has a simple structure to introduce fluorine-18 on the piperidine
ring via ethyl linker. EGFR tyrosine kinase inhibitors of HKI-272,
EKB-569 and BIBW2992 have a Michael acceptor group, which
plays a crucial role in inhibiting EGFR via irreversibly covalent
bond formation with cysteine residue.20–23 To the FTP2, Michael
addition unit was inserted. In order to evaluate the effectiveness
of Michael acceptor unit, FTP3 and FTP4 were designed in which
labeling sites are linked with two different alkyl chains. FTP5
was converted methylol unit in FTP2 to methyl group for simplifi-
cation of the synthetic route.
2.3. Radiosynthesis
[
18F]FTP2 was synthesized by two steps reaction using [18F]2-
fluoroethyl tosylate, which was prepared from 1,2-ditosylethane
in acetonitrile solution in the presence of Kryptofix222 and potas-
sium carbonate (Fig. 2a). The overall radiochemical yield and
radiochemical purity of [18F]FTP2 was 6.8%, and >99%, respec-
tively. Molar specific activity of [18F]FTP2 was 50 GBq/mmol at
minimum.
[
18F]FTP2 was incubated in sarin and blood serum for 1 h, but
no metabolized compounds were detected from HPLC analysis,
indicating [18F]FTP2 is stable in vitro condition (Fig. 2b).
Chemical synthetic procedures of these five non-radiolabeled
candidate compounds are summarized in Supporting information.
2.4. Cell uptake study
2.2. Evaluation of inhibitory potency of FTPs1-5 in vitro
NCI-H3255 and NCI-H1975 are human NSCLC cell lines express-
ing L858R mutated and L858R/T790M dual mutated EGFR, respec-
tively. To the cultures of these two NSCLC cells, [18F]FTP2 was
incubated, and amount of [18F]FTP2 uptake into the cell was
evaluated (Fig. 3a). Amount of up taken [18F]FTP2 into H3255
The inhibition ability of FTPs1-5 and gefitinib on EGFR tyrosine
kinase activity was evaluated using Promega ADP-Glo Kinase Assay
kit.24 IC50 values against wild type and three types of mutated
Fig. 1. Probe candidate compounds FTPs1–5 for L858R mutated epidermal growth factor receptor (EGFR) detection.
Table 1
Epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibition assay using Promega ADP-Glo Kinase Assay kit (n = 3).
Compound
EGFR kinase inhibition: IC50 value (nM). Numbers in parentheses are logIC50 (nM) standard error (n = 3)
WT
L858R
T790M
DM
(L858R/T790M)
FTP1
FTP2
FTP3
FTP4
FTP5
Gefitinib
2309 (3.363 0.316)
44 (1.643 0.094)
4370 (3.640 0.278)
2827 (3.451 0.105)
130 (2.114 0.186)
20 (1.301 0.056)
3414 (3.533 0.076)
9 (0.953 0.039)
2674 (3.427 0.266)
1835 (3.264 0.379)
27 (1.434 0.090)
21 (1.314 0.037)
Inactive
8966 (3.953 0.122)
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
7111 (3.852 0.103)
868 (2.938 0.047)
IC50 values greater than 10 mM are indicated as ‘‘inactive”.
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A. Makino et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Fig. 2. (a) Radiosynthesis of [18F]FTP2 and (b) HPLC chromatogram of (i) [18F]FTP2, (ii) [18F]FTP2 incubated in blood serum for 1 h, and (iii) [18F]FTP2 incubated in saline for
1 h. Y-axis indicates the relative amount of radioactivity contained in each fractions (0.50 mL/fractions).
Fig. 3. (a) Time courses of [18F]FTP2 uptake by H1975 and H3255 cells and (b) uptake of [18F]FTP2 after 30 min incubation with or without gefitinib (0.1–10 mM).
was reached to over 32.5 2.0%ID/mg protein after 60 min from
the incubation, which is three times higher than that into H1975
(9.51 0.6%ID/mg protein). Furthermore, the H3255 cell uptake
was decreased below half from the original by addition of 0.1–
10 mM gefitinib as inhibitor, whereas the H1975 was not changed,
indicating [18F]FTP2 uptake into H3255 is considered to be specific
binding to L858R mutated EGFR (Fig. 3b). Residual [18F]FTP2 into
H1975 and H3255 cells, which does not reduce by addition of
excess amount of inhibitors, show nonspecific binding.
dosage using animal PET/CT system (Fig. 4). Accumulation of
18F]FTP2 to H3255 cell transplanted region was observed, but
[
not to H1975. Biodistribution of [18F]FTP2 at 180 min from the
dosage is shown in Table 2. The accumulation of [18F]FTP2 in
H3255 tumor region was 2.5 0.3%ID/g, which is 2.7 times higher
than that in H1975 tumor region (0.9 0.1%ID/g). In organs other
than the transplanted tumor cells, similar 18F distribution was
observed. The high accumulation in the intestine is considered to
be caused by relatively hydrophobic structure of FTP2. [18F]FTP2
is trapped in the reticuloendothelial system such as liver, and then
transferred to the intestine via bile excretion. In case of H3255
bearing mice, tumor to blood, muscle and lung signal intensity
ratio was reached to 3.21, 5.50, and 2.53, respectively. On the other
hand, those value of H1975 bearing mice was 1.25, 2.00 and 0.90,
respectively.
2.5. In vivo imaging
[
18F]FTP2 was intravenously administrated to the BALB/c nude
mice subcutaneously transplanted with H3255 and H1975 cells,
and positron images were acquired during 180–200 min from the
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A. Makino et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 5. Effect of co-injection of gefitinib on biodistribution of [18F]FTP2 (3h post-
injection). The graphs show the mean %ID/g with error bars giving the standard
deviation (*P < 0.01).
3. Conclusion
Thienopyrimidine derivatives of FTPs1-5 were newly designed
and synthesized as new PET probe candidate compounds for detect-
ing gefitinib treatment sensitive NSCLC. FTP2 having Michael
acceptor unit in the structure showed high affinity (low IC50 value)
to gefitinib sensitive EGFR tyrosine kinase with L858R mutation but
high value to gefitinib resistant T790M mutation. The cell-uptake
and in vivo blocking study show that [18F]FTP2 binds specifically
to EGFR tyrosine kinase with gefitinib sensitive L858R mutation
but not to the gefitinib resistant L858R/T790M mutation. In vivo
study indicates [18F]FTP2 can clearly visualize H3255 NSCLC
expressing L858R mutated EGFR tyrosine kinase. [18F]FTP2 PET
study is useful to the patients undergoing molecular target therapy
with gefitinib. Accumulation of [18F]FTP2 indicates the NSCLC is
sensitive against gefitinib treatment, and the reduction helps early
detection of the occurrence of the EGFR secondary mutation, which
causes drug tolerance. Therefore, [18F]FTP2 is a potential candidate
compound for detecting gefitinib treatment sensitive NSCLC.
Fig. 4. PET-CT image of [18F]FTP2 in (a, b) H3255 (EGFR L858R, gefitinib positive)
and (c, d) H1975 (EGFR L858R/T790M gefitinib negative) bearing mice at 3 h post-
injection. Panels (a, c) and (b, d) show coronal and transverse images, respectively.
Table 2
Biodistribution of [18F]FTP2 in tumor bearing mice after 3 h from the dosage. The
values represent the mean SD (n = 5).
Tissue
H3255
H1975
(% ID/g)
EGFR (L858R)
EGFR
(L858R/T790M)
Blood
0.76 0.07
0.70 0.09
0.56 0.10
1.19 0.33
38.8 8.0
0.93 0.10
1.44 0.12
0.98 0.10
0.56 0.13
2.47 0.34
0.45 0.05
2.44 0.51
0.74 0.08
0.80 0.13
0.63 0.06
1.30 0.32
41.0 6.0
0.94 0.09
1.39 0.12
1.02 0.17
0.61 0.10
0.92 0.08
0.46 0.04
2.11 0.80
Spleen
Pancreas
Stomach
Intestine
Kidney
Liver
Lung
Heart
Tumor
Muscle
Bone
4. Experimental section
4.1. Instruments
Radiosynthesis was partly performed on JFE Hybrid synthesizer,
cassette-type multipurpose automatic synthesizer module (JFE
Engineering Corp., Tokyo, Japan). HPLC used was Prominence HPLC
system (Shimadzu Corp., Kyoto, Japan). Radioactivity was mea-
sured by using 1480 Wizard 3 auto gamma counter (PerkinElmer,
Waltham, MA, USA). PET scans were acquired by using a Triumph
(TriFoil Imaging Inc., Chatsworth, CA, USA).
Accumulation ratio
Tumor/Blood
Tumor/Muscle
Tumor/Lung
3.21
5.50
2.53
1.25
2.00
0.90
2.6. In vivo blocking study
4.2. Enzyme assay
[
18F]FTP2 was co-injected with excess amount of gefitinib (500
The inhibition ability of FTPs1-5 on EGFR tyrosine kinase activ-
ity was performed using an ADP-Glo Lipid Kinase Assay kit (Pro-
mega, USA). The assay was carried out according to the ADP-Glo
technical manual using purified recombinant EGFRs.
mM) to the H3255 or H1975 bearing mice, and radioactivity accu-
mulated at these tumor regions was evaluated by organ harvesting
method after three hours from the dosage. As shown in Fig. 5, the
[
18F]FTP2 accumulation in H3255 cells was significantly reduced
to the 37% from the control, but that in H1975 was not changed.
These results were well-correlated with in vitro cell uptake study
shown in Fig. 3, indicating [18F]FTP2 selectively binds to L858R
mutated EGFR.
4.3. Radiosynthesis
For the syntheses, commercially available reagents and solvents
were used without further purification. 2-[18F]Fluoroethyltosylate
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A. Makino et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
was synthesized using Hybrid synthesizer (JFE Engineering).
Briefly, [18F] fluoride solution containing 10 mg Kryptofix 2.2.2
(Sigma-Aldrich, St. Louis, USA) and 2.5 mg sodium carbonate
(Sigma-Aldrich) was heated at 120 °C and the solvent was com-
pletely removed by repeated addition of acetonitrile. To the reac-
tion vial, 1,2-bis(tosyloxy)ethane (20 mg) dissolved in acetonitrile
(0.80 mL) was added, and the mixture was heated at 100 °C for 8
min. After the solution was diluted by 0.80 mL water, 2-[18F]fluo-
roethyltosylate was isolated by COSMOSIL 5C18-AR-II column (10
mm I.D. Â 250 mm, Nacalai Tesque, Kyoto, Japan) by 50% acetoni-
trile aqueous solution as eluent (flow rate: 4.0 mL/min). Collected
solution was diluted with water (15 mL), loaded on Sep-Pak C18
column, and dried with nitrogen gas flow. Radioactivity eluted
from the Sep-Pak by acetonitrile (1.0 mL) was 6.4 Â 102 MBq, and
the total synthetic time was 69 min. Precursor (1.0 mg) dissolved
in acetonitrile (0.11 mL), DMF (0.04 mL) and triethylamine (0.01
mL) was added. The reaction mixture was heated at 110 °C for
25 min, and then purified by HPLC using COSMOSIL 5C18-AR-II col-
umn (10 mm I.D. Â 250 mm). Acetonitrile/water containing 0.1%
trifluoroacetic acid (v/v = 0 min: 15/85 ? 5 min: 30/70 ? 15 min:
35/65, flow rate: 5 mL/min) was used as eluent. The product was
evaporated to dryness and dissolved in PBS (À) containing 1%
DMSO to give [18F]FTP-2 (30 MBq). The total synthetic time (2
steps) was 158 min, and decay corrected yield was 6.8%. The radio-
chemical purity and specific activity of [18F]FTP-2 was over 99%
and 51.8 GBq/mmol, respectively.
by the Kyoto University and University of Fukui Animal Care Com-
mittees. Five-week-old male BALB/c nude mice were purchased
from Japan SLC, Inc. (Hamamatsu, Japan). Tumor-model mice were
prepared by subcutaneous injection of cells (1.0 Â 107 H3255 cells
or 3.0 Â 106 H1975 cells in the right shoulder) suspended in 100 mL
MatrigelTM (BD Biosciences, Franklin Lakes, NJ, USA). Approximately
4 weeks after implantation, the mice underwent tracer studies.
4.7. In vivo imaging and biodistribution study
On imaging, [18F]FTP-2 (9.1–14 MBq) in saline/0.5% Tween 80
was injected intravenously and PET scanning was started 3 h
post-injection. Dynamic PET images were acquired for 20 min.
The images were reconstructed using three-dimensional ordered
subset expectation maximization (3D-OSEM). Soon after the PET
imaging, the mouse were sacrificed and radioactivity in weighed
tumors, blood, muscle, and lungs were measured using a
c counter.
On biodistribution study, [18F]FTP-2 (0.185 MBq) was injected
intravenously into the tails of the tumor-bearing nude mice. For
the blocking study, the gefitinib solution adjusted final concentra-
tion to be 500 mM was co-injected with [18F]FTP-2. The mice were
sacrificed at 3 h post-injection. Tumors and organs of interest were
removed and weighed, and the radioactivity was measured.
Acknowledgments
[
18F]FTP-2 solution (1 mL) was mixed saline or blood serum
This work was supported by the Practical Research for Innova-
tive Cancer Control from the Japan Agency for Medical Research
and Development (AMED) and Grants-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science.
(100 mL), and incubated for 1 h. The mixture was centrifugated
after addition of methanol (200 mL). The recovery rate from the
blood serum was over 90%. The supernatant was analyzed by COS-
MOSIL 5C18-AR-II column (4.6 mm I.D. Â 150 mm) using acetoni-
trile/water containing 0.1% trifluoroacetic acid (v/v = 0 min:
20/80, 10 min: 30/70, flow rate: 1.0 mL/min) as eluent. The eluent
was fractionated every 0.5 mL, and radioactivity included in each
fractions against total one was measured.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.02.007.
4.4. Cell culture
References
H325525 and H1975, human derived lung adenocarcinoma,
were provided by Dr. Juri G.Gelovani’s lab., Dept. of Experimental
Diagnostic Imaging, MD Anderson Cancer Center, Houston TX, in
2007, were cultured in Dulbecco’s Modified Eagle Medium: Nutri-
ent Mixture F-12 (DMEM/F12) (ThermoFisher Scientific, Waltham,
MA, USA) supplemented with fetal bovine serum (FBS, 10% for
H1975 and 20% for H3255) (ThermoFisher Scientific, Waltham,
MA, USA) and 100 U/mL penicillin and 100 g/mL streptomycin
under 37 °C in a well-humidified incubator with 5% CO2 and 95%
ambient air.
1. Ono M, Hirata A, Kometani T, et al. Mol Cancer Ther. 2004;3:465–472.
2. Janku F, Garrido-Laguna I, Petruzelka LB, Stewart DJ, Kurzrock R. J Thorac Oncol.
2011;6:1601–1612.
3. Paez JG, Janne PA, Lee JC, et al. Science. 2004;304:1497–1500.
4. Rosell R, Carcereny E, Gervais R, et al. Lancet Oncol. 2012;13:239–246.
5. Kobayashi S, Boggon TJ, Dayaram T, et al. N Engl J Med. 2005;352:786–792.
6. Pao W, Miller VA, Politi KA, et al. PLoS Med. 2005;2:225–235.
7. Suda K, Mizuuchi H, Maehara Y, Mitsudomi T. Cancer Metastasis Rev.
2012;31:807–814.
8. Goss G, Tsai CM, Shepherd FA, et al. Lancet Oncol. 2016;17:1643–1652.
9. Mok TS, Wu YL, Ahn MJ, et al. N Engl J Med. 2017;376:629–640.
10. Liao BC, Lin CC, Lee JH, Yang JCH. Lung Cancer. 2017;110:7–13.
11. Bahce I, Smit EF, Lubberink M, et al. Clin Cancer Res. 2013;19:183–193.
12. Memon AA, Jakobsen S, Dagnaes-Hansen F, Sorensen BS, Keiding S, Nexo E.
Cancer Res. 2009;69:873–878.
4.5. Cell uptake study
13. Seimbille Y, Phelps ME, Czernin J, Silverman DHS. J Labelled Compd Radiopharm.
2005;48:829–843.
14. Traxl A, Beikbaghban T, Wanek T, et al. Nucl Med Biol. 2017;52:7–15.
15. Kimura H, Okuda H, Ishiguro M, et al. ACS Med Chem Lett. 2017;8:418–422.
16. Bugge S, Buene AF, Jurisch-Yaksi N, et al. Eur J Med Chem. 2016;107:255–274.
17. Bugge S, Kaspersen SJ, Larsen S, et al. Eur J Med Chem. 2014;75:354–374.
H1975 and H3255 NSCLC cells were cultured to confluence in
24 well plates. Cells were incubated in FBS-free DMEM/F12 med-
ium containing [18F]FTP-2 (0.60 MBq/mL) with or without gefitinib
(0.1–10 mM) for 30 min, and then washed three times with PBS (À)
containing 1.0% DMSO. The cells were dissolved by 0.2 M sodium
hydroxide solution, and the radioactivity was measured by auto-
matic gamma counter. The protein concentration was measured
using Pierce BCA Protein Assay kit (ThermoFisher Scientific, Wal-
tham, MA, USA) and used for normalization.
18. Pedeboscq S, Gravier D, Casadebaig F, et al. Eur
J
Med Chem.
2010;45:2473–2479.
19. Waterson AG, Petrov KG, Hornberger KR, et al. Bioorg Med Chem Lett.
2009;19:1332–1336.
20. Kwak EL, Sordella R, Bell DW, et al. Proc Natl Acad Sci USA.
2005;102:7665–7670.
21. Li D, Ambrogio L, Shimamura T, et al. Oncogene. 2008;27:4702–4711.
22. Tsou HR, Overbeek-Klumpers EG, Hallett WA, et al.
2005;48:1107–1131.
J Med Chem.
23. Wu CH, Coumar MS, Chu CY, et al. J Med Chem. 2010;53:7316–7326.
24. Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA. Assay Drug Dev Technol.
2009;7:560–572.
4.6. Tumor model
Animal studies were conducted in accordance with our institu-
tional guidelines, and the experimental procedures were approved
25. Nishii R, Volgin AY, Mawlawi O, et al. Eur
J Nucl Med Mol Imaging.
2008;35:990–998.
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