Improved radiosynthesis of the apoptosis marker 18F-ICMT11 including biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2011.10.001

We improved the specific radioactivity of the apoptosis imaging isatin derivative ¹⁸F-ICMT11. We then evaluated ¹⁸F-ICMT11 in EL4 tumor-bearing mice 24 h after treatment with etoposide/cyclophosphamide combination therapy. Dynamic PE

Full text of DOI:10.1016/j.bmcl.2011.10.001

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6945–6949
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improved radiosynthesis of the apoptosis marker 18F-ICMT11 including
biological evaluation
Matthias Glaser a, , Julian Goggi b, , Graham Smith c, Matthew Morrison b, Sajinder K. Luthra a,
⇑
Edward Robins a, , Eric O. Aboagye c
a MDx Discovery (Part of GE Healthcare), Hammersmith Imanet Ltd, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom
b MDx Discovery (Part of GE Healthcare), The Grove Centre, White Lion Road, Amersham HP7 9LL, United Kingdom
c Comprehensive Cancer Imaging Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom
a r t i c l e i n f o
a b s t r a c t
We improved the specific radioactivity of the apoptosis imaging isatin derivative 18F-ICMT11. We then
evaluated 18F-ICMT11 in EL4 tumor-bearing mice 24 h after treatment with etoposide/cyclophosphamide
combination therapy. Dynamic PET imaging demonstrated increased uptake in the drug-treated
(0.115 0.011 SUV) compared to the vehicle-treated EL4 tumors (0.083 0.008 SUV). This effect
correlated to the observed increases in apoptotic index.
Article history:
Received 25 August 2011
Revised 29 September 2011
Accepted 3 October 2011
Available online 8 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Apoptosis
PET
Isatin
Caspase
Fluorine-18
Click chemistry
Apoptosis – programmed cell death – can be triggered both by
intrinsic and extrinsic cell signals and is fundamental to the mech-
anism of action of current cancer treatments.1 Ideally, a noninva-
sive molecular imaging agent that targets apoptosis should be
able to monitor early tumor response to classical anti-cancer ther-
apy. This would then support further decisions on treatment op-
tions for patients. An apoptosis tracer would be also beneficial in
preclinical development of novel chemotherapeutic agents.
association with immunohistochemical measures of apoptosis.19
In addition, there are indications that the apoptotic display of PS
can be reversible20 and can also occur during myotube formation.21
Imaging anionic phospholipids via radiolabelled c-carboxyglutamic
acid (Gla),22 duramycin,23 or small peptide-based radiotracers24
have been suggested as an alternative approaches. However, it ap-
pears likely that some of the above mentioned limitations of annex-
in-V still apply.
So far, various avenues have been explored to investigate apopto-
sis in the living organism. A good overview of the field can be gath-
ered from several well-written review articles.1–4 Numerous studies
have been dedicated to annexin-V, a calcium dependent phosphati-
dyl serine (PS) binding protein of 32–36 kDa.3 The technetium-99 m
labelled annexin-V probe is probably the most advanced clinical
apoptosis tracer for single photon computed tomography (SPECT).
Annexin-V has been also conjugated with PET radionuclides such
as 18F and 124I.5–16 There are however known limitations to this vec-
tor protein. The radiotracer is not necessarily able to differentiate
between necrosis and apoptosis as in both cases PS residues are ex-
posed.17,18 Recent data from a mouse breast tumor model pointed to
potential problems of 99mTc-annexin-V indicating absence of an
Imaging of cysteine aspartate-specific protease (caspase) activity
in the apoptotic signaling pathway has been identified as an alterna-
tive target to visualize cell death. A number of 5-pyrrolidinylsulfonyl
isatin derivatives were confirmed to bind with high affinity to the
most relevant executioner caspase-3.25 These compounds demon-
strated better cell permeability compared to the Z-VAD-fmk pep-
tide26 to reach activated caspase.
Figure 1 shows the structures of published 18F/11C labelled isatin
compounds 18F-WC-II-89,27 18F-WC-IV-3,28 18F-WC-98,28 and 18F-
1.29 We recently reported a Click chemistry strategy to synthesize
18F-ICMT11.30 This approach avoided protection of the reactive
dicarbonyl function. However, that process gave rise to a stable
by-product which could not be removed from the 18F tracer. Since
this material might potentially compete in the caspase-3 binding,
we undertook further optimization of the radiochemistry protocol
to address that issue. We also investigated the in vivo behavior of
18F-ICMT11 – as prepared by the new protocol – in the murine lym-
phoma xenograft EL4 model.31
⇑
Corresponding author. Tel.: +44 149 454 6121; fax +44 149 454 3284.
E-mail address: matthias.glaser@ge.com (M. Glaser).
Present address: Singapore BioImaging Consortium, HELIOS, 11 Biopolis Way,
Singapore 138667, Singapore.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.001

6946
M. Glaser et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6945–6949
O
N
O
O
O
N
O
O
S
S
O
O
N
N
11CH3
O
O
O
O
18F
[18F]WC-98
[18F]WC-II-89
O
O
O
O
O
O
S
N
S
O
N
O
N
N
18F
O
O
O
OH
18F
[18F]-1
[18F]WC-IV-3
O
O
O
S
N
O
N
N
N
N
O
F
18F
F
[18F]ICMT-11
Figure 1. Structures of isatin analogs labelled with positron emitters.
18F-ICMT11 has been prepared for the initial in vivo study
following the original radiochemistry protocol.30 The estimated
specific radioactivity, impurity level and radiochemical yields are
given in Table 1 (further information on the radiochemical meth-
ods can be found in the Supplementary data). The protocol could
be significantly improved by introducing bathophenanthroline
disulfonic acid disodium ligand (BPDS) as an additive to stabilize
the Cu(I) catalyst (Route A, Scheme 1). We found that BPDS helped
both to reduce the amount of required alkyne precursor 2 (1 mg vs
3 mg), and also to shorten the reaction time (15 min vs 30 min).
The resulting formulated 18F-ICMT11 showed a reduced carrier
level (Table 1). While our study was in progress, other investigators
also discovered that BPDS accelerates the kinetics of the
cycloaddition.32,33
The radiosynthesis of 18F-ICMT11 following Route A gave rise to
a stable by-product of almost identical HPLC retention time as the
radioactive product. It was possible to isolate a small quantity of
that material (<100 l
g). However, MS and 1H NMR data were not
conclusive. Accurate MALDI-TOF and ESI MS suggested two species
(see Supplementary data, Fig. 4). The UV spectrum of the material
revealed a profile similar to the stable 18F-ICMT11 reference sam-
ple. For this reason, it appeared particularly important to keep the
Table 1
Comparison of specific radioactivities and radiochemical yields of 18F-ICMT11.
Route
Method
Specific Radioactivitya (GBq/
lmol)
Amount of stable producta
(l
g/mL)
EOS RCYb
A
A
B
B
B
Literature30
Ic,d
1.2 (1)
13.8 (1)
3.4 1.5% (5)
3.2 0.8% (5)
7.0 5.6 (5)
13.3 4.4 (3)
17 3 (3)
24 19 (3)
4.6 4.3 (5)
4.8 3.4 (3)
1.6 0.9 (3)
4.1 4.1 (3)
IIc,e
3
1% (3)
IIIc,f
1% (2)
3.0 2.6% (3)
IVc,g
The number of experiments is given in brackets.
a
Estimated, based on isatin-like stable by-product.
b
End of synthesis radiochemical yield of formulated product, nondecay corrected, referring to [18F]-fluoride.
Using BPDS in the cycloaddition step.
c
d
e
f
Using isatin precursor 2, without carbonyl protecting group.
HCl/microwave deprotection of intermediate 4.
H2SO4 deprotection of intermediate 4.
g
C18-SepPak purification prior to H2SO4 deprotection of intermediate 4.

M. Glaser et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6945–6949
6947
Route A
i
O
N
O
O
O
N
O
O
S
S
O
O
N
N
N
N
N
O
O
F
18F
F
F
F
2
[18F]ICMT-11
iii
Route B
ii
O
O
N
O
O
O
O
S
O
O
S
O
N
O
N
N
N
N
N
O
O
F
F
F
18F
F
4
3
Scheme 1. Preparation of 18F-ICMT11. (i) 2-[18F]fluoroethylazide, CuSO4/Na-ascorbate, pH 5.0, BPDS, 30 min rt, preparative HPLC; (ii) 2-[18F]fluoroethylazide, CuSO4/Na-
ascorbate, pH 5.0, BPDS, 15 min rt; (iii) 6 N HCl, microwave (3 Â 2s @ 50 W), preparative HPLC.
level of by-product as low as possible in order to avoid a potential
caspase-3/7 blocking.
lymphoma.35 That study found up to twofold increased tumor
binding after drug treatment.
From studies with model isatin compounds we concluded that
the level of stable impurity did not depend on the presence of the
sulfonamide moiety. Also, protecting a sulfonamide-free model isat-
in with a cyclic acetal34 suppressed the impurity (data not shown).
Therefore, we prepared an acetal protected isatin alkyne precursor
3 for the Click labelling chemistry (Route B). The resulting interme-
diate 4 was formed in quantitative radiochemical yields. The depro-
tection was achieved by using both microwave heating (HCl
additive, Method II, used to prepare samples for biology study) or
conventional heating (H2SO4 additive, Method III).
The experimental results revealed that the radiosynthesis of
18F-ICMT11 had been improved with regard to labelling efficiency.
However, the improvements in the specific radioactivity from
Routes A and B were somewhat lower than anticipated. It appeared
that any isatin alkyne 2 – either as nonprotected precursor or
in situ formed upon acidic deprotection – would react with Cu(I)
species to form the unwanted carrier material. Therefore an exper-
iment was designed to investigate the removal of Cu(I) prior to
deprotection in Route B (Method IV). However, the by-product
was still observed, albeit to a lesser extent. This finding suggested
that the by-product formation was either independent of copper
catalysis or the pre-deprotection purification was inefficient in
removing reactive copper species.
Finally, a promising one-pot approach was investigated with
the aim to increase the overall radiochemical yield and to simplify
the process (Method V). Here, 18F-ICMT11 was prepared in the
same vial that was used to produce 2-[18F]fluoroethylazide. With-
out distillation of 2-[18F]fluoroethylazide, 18F-ICMT11 could be ob-
tained using copper wire as Cu(I) source. The analytical data are
shown in the Supplementary data. However, the protocol was
not further developed.
The biology studies described here were performed in order to
investigate the performance of 18F-ICMT11 for imaging combina-
tion etoposide and cyclophosphamide therapy-induced apoptosis
in an EL-4 (murine lymphoma) tumor model in vivo using PET.
The isatin compound 18F-ICMT11 has been previously evaluated
for cyclophosphamide-induced tumor apoptosis in 38C13 murine
Literature studies have shown that a murine lymphoma (EL4) tu-
mor model undergoes large-scale apoptosis following treatment
with etoposide.31,36 EL4 cells form a solid tumor when implanted
subcutaneously into C57/BL6 mice and readily undergo apoptosis
in vivo when exposed to chemotherapeutic agents. It has been dem-
onstrated that treatment of tumor bearing mice with a combination
of cyclophosphamide and etoposide for 48 h show an increase in the
average levels of apoptosis in EL4 tumors from basal levels of 4% to
32%.31 The distribution of apoptosis was heterogenous, with regions
of some tumor sections containing large numbers of apoptotic
cells.31 Additional in-house validation studies confirmed that a sim-
ilar level of apoptosis can be seen 20–24 h post therapy in the EL4 tu-
mor model (see Supplementary data, Table 1). Additionally, use of
animals 24 h post treatment was logistically and ethically preferred
to using animals at the 48 h time point. Therefore, 24 h was chosen
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0
10
20
30
40
50
60
70
Time (minutes)
Pre treatment tumour
Post treatment tumour
Figure 2. Summary of imaging data of the lower specific activity compound 18F-
ICMT11 in the EL4 model. Data expressed dynamically prior to treatment and post
treatment with combined etoposide and cyclophosphamide therapy (SUV SD,
n = 5 pre-treatment ⁄one animal data omitted due to reconstruction issues, n = 3
post treatment).

6948
M. Glaser et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6945–6949
700000
600000
500000
400000
300000
200000
100000
0
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0
10 20 30 40 50 60 70 80
Time (minutes)
Pre treatment tumour
Post treatment tumour
Post vehicle tumour
Figure 3. Summary of imaging data of the high specific activity compound 18F-
ICMT11-hsp in the EL4 model. Data expressed dynamically prior to treatment
(n = 3) and post treatment with either vehicle (n = 1) or combined etoposide and
cyclophosphamide therapy (n = 2) (SUV SD).
Figure 4. Apoptosis levels (arbitrary units: assessed using the Glo assay for
activated caspase 3) in tumors treated with vehicle or tumor treated with anti-
cancer therapy (combination of etoposide and cyclophosphamide) displaying
significantly increased levels of caspase-3 in the drug treated tumors.
as the optimum timepoint for apoptosis post administration of eto-
poside/cyclophosphamide.
BPDS additive increased the labelling efficiency of the 2-[18F]fluo-
roethylazide cycloaddition step. Acetal protection of the reactive
isatin carbonyl group during that reaction did not completely sup-
press the formation of a stable by-product.
The data presented here demonstrated that the EL4 therapy
model was suitable for imaging apoptosis and assessment of both
18F-ICMT11 and 18F-ICMT11-hsp.
Here, the acquired 18F-ICMT11 images demonstrate uptake into
EL4 tumors pre-treatment of 0.058 0.013 SUV at 40–60 min post
injection, see Figure 2. Twenty-four hours following vehicle treat-
ment, the uptake into the vehicle treated tumors was similar
(0.048 0.015 SUV at 40–60 min p.i.), whereas 24 h after anti-can-
cer treatment (combined etoposide and cyclophosphamide therapy)
the uptake into the treated tumors was significantly increased to
0.085 0.019 SUV at 40–60 min p.i. see Supplementary data, Table
2, for full details).
Dynamic imaging over a 1 h period with both 18F-ICMT11 and
18F-ICMT11-hsp in the EL4 tumor model pre and post therapy (in-
tra animal longitudinal imaging studies) was successfully accom-
plished. Overall, the dynamic time versus radioactivity data
showed significantly improved uptake in the treated versus non-
treated EL4 tumors that was associated with the observed in-
creases in apoptotic index as determined using the Caspase Glo
assay (for details see Supplementary data).
These results demonstrate that tumor uptake was significantly
different in the murine tumors pre and post therapy, indicating
that changes in the level of apoptosis post therapy can be detected
using the imaging agent 18F-ICMT11. For comparison, region of
interest (ROI) analysis of skeletal muscle demonstrated that uptake
was equivalent pre and post therapy (0.122 0.07 SUV and
0.138 0.11 SUV).
In order to assess the utility of the high specific activity com-
pound, 18F-ICMT11-hsp, radiotracer uptake was measured using
positron emission tomography in the same EL4 tumor bearing ani-
mals before treatment and following treatment with a combina-
tion of etoposide (67 mg/kg) and cyclophosphamide (100 mg/kg).
The acquired 18F-ICMT11-hsp images demonstrated uptake into
EL4 tumors pre-treatment of 0.094 0.009 SUV at 40–60 min post
injection, see Figure 3. Twenty-four hours following vehicle treat-
ment, the uptake into the vehicle treated tumors was not statisti-
cally different (0.083 0.008 SUV at 40–60 min p.i.), whereas 24 h
after anti-cancer treatment (combined etoposide and cyclophos-
phamide therapy) the uptake into the treated tumors was signifi-
cantly increased to 0.115 0.011 SUV at 40–60 min p.i. see
Supplementary data, Table 2, for full details).
The conclusion from this series of imaging studies is that 18F-
ICMT11 shows promise as a PET agent to measure the levels of
apoptosis in treated tumors. The improved specific activity com-
pound 18F-ICMT11-hsp displayed marginally higher tumor reten-
tion than the lower specific activity compound 18F-ICMT11 with
no significant difference in uptake observed in the reference tissue,
skeletal muscle.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.001.
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