Synthesis and in vitro evaluation of 68Ga-DOTA-4-FBn-TN14003, a novel tracer for the imaging of CXCR4 expression

Article abstract of DOI:10.1016/j.bmc.2011.12.052

The expression of the chemokine receptor CXCR4 in tumors is associated with tumor aggressiveness and poor prognosis for the patient and contributes to metastatic seeding. Therefore it is of high interest to find a specific PET tracer for the imaging of CX

Full text of DOI:10.1016/j.bmc.2011.12.052

Bioorganic & Medicinal Chemistry 20 (2012) 1502–1510
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of 68Ga-DOTA-4-FBn-TN14003, a novel
tracer for the imaging of CXCR4 expression
Ute Hennrich a, , Lisa Seyler a, Martin Schäfer b, Ulrike Bauder-Wüst b, Michael Eisenhut b,
⇑
Wolfhard Semmler a, Tobias Bäuerle a
a Department of Medical Physics in Radiology, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
b Department of Radiopharmaceutical Chemistry, DKFZ, Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2011
Revised 23 December 2011
Accepted 23 December 2011
Available online 2 January 2012
The expression of the chemokine receptor CXCR4 in tumors is associated with tumor aggressiveness and
poor prognosis for the patient and contributes to metastatic seeding. Therefore it is of high interest to find
a specific PET tracer for the imaging of CXCR4 expression in tumors. The aim of this study was the syn-
thesis, 68Ga labeling and first evaluation of DOTA-4-FBn-TN14003 as a potential PET tracer for this pur-
pose. DOTA-4-FBn-TN14003 was synthesized using solid phase peptide synthesis and radiolabeling of
this versatile precursor was performed with 68Ga, which was obtained from a 68Ge/68Ga generator.
68Ga-DOTA-4-FBn-TN14003 was reproducibly obtained in isolated radiochemical yields of 72.5 4.9%
Keywords:
Molecular imaging
CXCR4
PET
Gallium-68
with an excellent radiochemical purity of >99.5%. Specific activities of up to 29.8 3.1 GBq/
achieved. In competition binding assays with SDF-1 , human T cell lymphoma Jurkat cells expressed high
levels of CXCR4 whereas human breast cancer MDA-MB-231 cells expressed significantly lower levels of
this chemokine receptor. The inhibition constants (IC50 of Ga-DOTA-4-FBn-TN14003 and
4-FBn-TN14003 to CXCR4 were determined in a competition assay against 125I-SDF-1
using Jurkat as
lmol were
a
)
a
well as MDA-MB-231 cells. The IC50 values of Ga-DOTA-4-FBn-TN14003 (1.99 0.31 nM) and 4-FBn-
TN14003 (4.07 1.00 nM) proved to be comparable, indicating negligible influence of the metal complex.
These results suggest 68Ga-DOTA-4-FBn-TN14003 as a promising agent for the imaging of CXCR4 expres-
sion in tumors and metastases.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cancer.7,8 Consequently, CXCR4 is a potential imaging target for
the diagnosis, staging and therapeutic monitoring of metastases.
Chemokines play a critical role in tumor progression and metas-
tasis. In this regard, the chemokine receptor CXCR4 is expressed by
tumor cells including breast, prostate and lung cancer that are
metastasizing to the respective target organs.1–3 In breast cancer,
overexpression of CXCR4 predicts poor clinical outcome and is clo-
sely associated with lymph node metastasis.4,5 During the develop-
ment of metastasis, CXCR4 interacts with its ligand CXCL12
Imaging of tumors and in particular metastases is currently lim-
ited to assessing morphology and metabolic activity by magnetic
resonance imaging (MRI), computed tomography (CT), scintigra-
phy, single photon emission computed tomography (SPECT) or
positron emission tomography (PET). Use of these techniques,
however, results in a relatively late detection of disseminated
lesions, that is, after tumors or macrometastases in the range of
several millimeters have developed. For this reason, specific imag-
ing of molecular targets expressed by disseminated tumor cells
and micrometastases is attractive for early diagnosis. In this con-
(stromal cell-derived factor-1, SDF-1
a
) which is expressed in liver,
to
lung, lymph nodes, bone and other tissues.6 Binding of SDF-1
a
CXCR4 results in a variety of responses such as chemotaxis as well
as cell survival, migration and proliferation. The expression of
text, we recently presented the integrins
avb3 and avb5 as molec-
SDF-1
colonization of disseminated tumor cells that express CXCR4. Inhi-
bition of the CXCR4-SDF-1 axis was reported to reduce orthotopic
a by these organs is supposed to be crucial for homing and
ular targets for the imaging of bone metastases.9
Several approaches have been performed to image CXCR4
expression in tumor models using 111In-labeled peptides and
125I-labeled antibodies by SPECT.10–12 Recently imaging of CXCR4
by PET was described, for example, Nimmagadda et al. reported
the feasibility of imaging CXCR4 by two different 64Cu-labeled
PET tracers in orthotopic and metastatic breast cancer in mice13
as well as a subcutaneous brain tumor model.14 Here, we describe
a
and metastatic tumor growth in experimental models of breast
⇑
Corresponding author. Tel.: +49 6221 422686; fax: +49 6221 422585.
E-mail address: u.hennrich@dkfz.de (U. Hennrich).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.052

U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
1503
the synthesis, radiolabeling and evaluation of 68Ga-DOTA-4-FBn-
TN14003 as a novel tracer for the imaging of CXCR4 expression
in MDA-MB-231 human breast cancer and Jurkat human
lymphocyte cell lines as well as the expression levels of the recep-
tor on both cell lines.
radioactive samples were either counted in the
(Packard Canberra, Minnesota, USA) or the Wizard
c
-counter Cobra II
2 (2480
T
automatic gamma counter; Perkin–Elmer, Rodgau, Germany).
Analysis of the radiolabeled compound was performed using
analytical reversed-phase high performance liquid chromatogra-
phy (RP-HPLC). The HPLC system (system I) contained a L6200
pump from Merck-Hitachi (Darmstadt, Germany), a variable wave-
length UV detector (254 nm) from Latek (Heidelberg, Germany)
and a NaI(Tl) gamma detector from Bioscan (Washington, USA).
Analytical HPLC was performed using a Chromolith Performance
RP-18e column (100 ꢀ 4.6 mm; Merck, Darmstadt, Germany). The
solvent gradient was raised from 0% to 100% methanol in 5 min
at a flow rate of 4.0 mL/min. As aqueous phase water containing
0.1% TFA was used. Purification of the non-radioactive peptides
was performed by semi-preparative reversed-phase HPLC using
the following system (system II) from Dionex (Idstein, Germany):
an Ultimate 3000 LPG-3400A pump, a variable four wavelength
Ultimate 3000 VWD-3400RS UV/VIS detector (222, 254, 280 nm),
analysis of HPLC data was performed with Chromeleon 6.80 soft-
2. Materials and methods
2.1. Materials
All commercially available chemicals were of analytical grade or
better and used without further purification unless otherwise
noted. The main suppliers were Sigma–Aldrich (Taufkirchen,
Germany) and Merck (Darmstadt, Germany). Tris-tBu-DOTA was
synthesized modifying a three step procedure previously described
by Wängler et al.15 The last reaction step, the debenzylation, was
not performed using hydrogen gas as described but by generating
hydrogen in situ from ammonium formate in the presence of palla-
dium black in absolute methanol at 60 °C.16 68Ga was obtained from
a self-assembled 68Ge/68Ga generator based on a pyrogallol resin
support.17 Typically, about 500 MBq of 68Ga (t1/2 = 68 min) were
eluted with 5.5 M HCl and trapped on a small anion-exchanger
column (AG1X8, Biorad, Richmond, CA, USA). The activity was
eluted from this cartridge using ultrapure water (Merck, Darmstadt,
ware. As column
a Chromolith Performance RP-18e column
(100 ꢀ 10 mm; Merck, Darmstadt, Germany) was used. The solvent
gradient was raised from 5% to 100% acetonitrile (a) or methanol
(b) in 5 min at a flow rate of 6 mL/min. The aqueous phase con-
sisted of water containing 0.1% TFA. MALDI-TOF-MS was per-
formed on a microflex LT (Bruker Daltonics, Bremen, Germany).
Germany) in a final volume of 300 lL. For the cell assays, the
(a)
Arg1
O
O
Nal3
O
Tyr5
O
Lys7
O
H
N
H
N
H
N
H
N
D-Lys8
N
H
N
H
N
H
N
H
O
Arg2
O
O
Arg6
F
S
Cys4
O
O
Cys13
S
O
Arg11 O
N
H
H
H
N
N
N
H2N
N
H
N
H
Pro9
Arg14
O
Cit12
O
Tyr10 O
1
O
O
OH
(b)
N
O
68Ga
N
N
N
O
NH
O
O
O
Arg1
O
O
Nal3
O
Tyr5
Lys7
H
N
H
N
H
N
H
N
N
H
N
H
N
H
N
H
D-Lys8
O
O
Arg2
O
O
O
Arg6
O
F
S
Cys4
O
Cys13
S
Arg11
O
N
H
H
H
N
N
N
H2N
N
H
N
H
Pro9
Arg14
O
Cit12
O
Tyr10 O
2
Figure 1. Structures of 4-F-Bn-TN14003 (1) and 68Ga-DOTA-4-FBn-TN14003 (2).

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U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
2.2. Synthesis of DOTA-4-FBn-TN14003 and 4-FBn-TN14003
were again purified by semi-preparative HPLC, now using system IIb
(tR = 3.31 min). The identity of 4-FBn-TN14003 was confirmed by
MALDI-TOF-MS (m/z calcd for C97H145FN33O19S2 ([M+H]+): 2160.5
found 2160.5).
The synthesis scheme of DOTA-4-FBn-TN14003 is shown in
Figure 2. The protected peptidyl resin was manually constructed
using Fmoc-based solid phase synthesis on Rink Amide resin
(0.71 mmol/g, 0.1 mmol scale). Fmoc-protected amino acid deriva-
tives (4 equiv) were successively condensed, using HBTU
(3.92 equiv) in the presence of DIPEA (4 equiv) for activation, in
DMF, allowing a reaction time of 60–90 min for each amino acid.
For side-chain protection the following groups were used: Pbf for
2.3. 68Ga-Labeling of DOTA-4-FBn-TN14003
To the [68Ga]Ga3+ eluate (200–500 MBq) in a polypropylene
screwcap vial HEPES solution (4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid, 5.5 M in ultrapure water, 110
as well as sodium hydroxide solution (30%, 15 L) and DOTA-4-
FBn-TN14003 (5 or 10 nmol in 5 or 10 L of ultrapure water,
lL) was added
Arg, Acm for Cys, tBu for Tyr, Boc for Lys, and Mtt for
D-Lys. Fmoc-
l
deprotection was performed by treatment of the resin with 50%
(v/v) piperidine-DMF for2 and 5 min. Foracylationof theN-terminal
amino group the resin was reacted for 90 min with 4-fluorobenzoic
acid (4 equiv) under HBTU activation as described above. The forma-
tion of the disulfide bridge between Cys4 and Cys13 was achieved by
reactingtheresinwiththallium(III)trifluoroacetate(4 equiv, techni-
cal grade) in the presence of anisole (5%) in DMF for 5 h. For the con-
l
respectively). The vial was heated for 10 min at 80 °C. Depending
on the application, the compound was either diluted to the desired
concentration (cell assays) or further purified by solid phase
extraction (stability studies). For purification, the reaction mixture
was diluted with water (1 mL) and passed over an Oasis HLB 1 cc
cartridge (Waters, Eschborn, Germany). Elution of the product
was achieved with absolute ethanol (0.8 mL). In order to perform
stability studies with the radiotracer, the solvent was evaporated
at 80 °C under a gentle stream of air and the residue was taken
up in phosphate buffered saline (PBS). Depending on the synthesis
conditions for 68Ga-DOTA-4-FBn-TN14003, specific activities of up
jugation of tris-tBu-DOTA, the
e-amino function of D-Lys8 was
selectively deprotected by treatment of the resin with 1.5% TFA
and 3% TIS (triisopropylsilane) in DCM for 3 ꢀ 30 min, followed by
washing of the resin with 10% DIPEA in DMF (5 ꢀ 5 min). Then the
reaction with tris-tBu-DOTA (4 equiv), activated as described above,
was performed for 3 h. Cleavage from the resin as well as side-chain
deprotection of the amino acid derivatives was achieved by treat-
ment with 95% TFA, 2.5% TIS and 2.5% water for 4 h. The crude pep-
tide was precipitated by dropwise addition of the reaction mixture
to ice cold diethyl ether and collected by filtration followed by wash-
ing with ice cold diethyl ether. Purification of DOTA-4-FBn-TN14003
was performed by semi-preparative HPLC (system IIa) and it was ob-
tained as a fluffy white powder after freeze drying the collected
product fractions (tR = 2.43 min). The identity of the peptide was
confirmed by MALDI-TOF-MS (m/z calcd for C113H171FN37O26S2
([M+H]+): 2546.9 found 2546.8) and NMR spectroscopy (see Supple-
mentary data). The parent peptide 4-FBn-TN14003 was synthesized
accordingly, omittingthe step of DOTA-conjugation. It was also puri-
fied by semi-preparative HPLC (tR = 2.41 min, system IIa). Because it
was not pure after the first HPLC purification, the collected fractions
to 29.3 3.2 GBq/lmol were achieved at the end of synthesis. The
radiochemical purity was analyzed by RP-HPLC (tR = 3.29 min; sys-
tem I) and the identity of the labeled peptide verified by co-elution
with the non-radioactive reference compound.
2.4. Synthesis of Ga-DOTA-4-FBn-TN14003
The non-radioactive standard compound Ga-DOTA-4-FBn-
TN14003 was obtained by reacting the peptide precursor (5 nmol
in 5
equivalents of a 3 mM solution of Ga(NO3)3 hydrate (in 0.1 M
hydrochloric acid) in the presence of 2.1 M HEPES buffer (10 L)
and 1 M hydrochloric acid (2 L). The pH value of the solution
lL ultrapure water and 35 lL 0.1 M HEPES buffer) with 4
l
l
was between 4.1 and 4.4 and the mixture was heated at 80 °C
for 10 min. After cooling, the pH value was adjusted to 7.4 by the
Acm tBu
Boc Mtt
tBu
Acm
Pbf
Pbf Pbf
Pbf
(a)
(b)
(c)
Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg
Acm tBu
Boc Mtt
tBu
Acm
Pbf
Pbf Pbf
Pbf
4-FBn-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg
tBu
Boc Mtt
tBu
Pbf
Pbf Pbf
Pbf
4-FBn-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg
tBu
Boc
tBu
Pbf
Pbf Pbf
Pbf
(d)
(e)
4-FBn-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg
tris-tBu-DOTA
4-FBn-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg
DOTA
NH2
Figure 2. Synthesis of DOTA-4-FBn-TN14003. Reagents: (a) stepwise elongation on Rink Amide resin; (b) 4-fluorobenzoic acid, HBTU, DIPEA; (c) Thallium(III) triflouroacetate,
anisole; (d) (1) 1.5% TFA and 3% TIS in dichloromethane, (2) tris-tBu-DOTA, HBTU, DIPEA; (e) TFA, TIS, water.

U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
1505
addition of 1 M sodium hydroxide solution and the compound was
used without further purification. The identity of the reference
compound was confirmed by MALDI-TOF-MS (m/z calcd for
Plot 11.0 and a sigmoidal dose-response fit. For comparison, the
experiments were also performed with 4-FBn-TN14003 instead
of Ga-DOTA-4-FBn-TN14003 as described above. For the respective
binding assays using MDA-MB-231 cells, the cells were trypsini-
zed, resuspended in the binding buffer and the assay was con-
ducted as described above.
C
113H169FGaN37O26S2 ([M+H]+): 2613.6 found 2613.3).
2.5. Stability of 68Ga-DOTA-4-FBn-TN14003
For the determination of the number of receptors per cell on the
different cell lines (Jurkat and MDA-MB-231) the assay was per-
The stability of 68Ga-DOTA-4-FBn-TN14003 was evaluated in
PBS as well as human plasma. For the stability in PBS, the radio-
tracer was incubated at 37 °C and its stability repeatedly tested
by analytical HPLC (system I). Human plasma (Sigma–Aldrich)
was incubated with the radiotracer at 37 °C and the stability of
the compound was tested after 30 and 90 min (n = 2, respectively).
formed using human SDF-1a (Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany) as inhibitor under the same assay conditions
as described above (n = 3–5). The Bmax calculation was conducted
using a competitive binding fit from GraphPad Prism 5.0 Software
(San Diego, California, USA).
Plasma samples (100 lL) were vortexed with an equal amount of
2.8. Cell binding assays using 68Ga-DOTA-4-FBn-TN14003
acetonitrile and centrifuged at 4 °C for 2 min at 13,000 rpm and
an aliquot of the supernatant solution was analyzed by HPLC (sys-
tem I). An aliquot of the plasma itself was analyzed by size exclu-
sion chromatography (SEC; Superdex 75, 0.4 mL/min, 0.1 M
phosphate buffer (pH 7.4) containing 500 mM arginine).
For the determination of uptake kinetics as well as internaliza-
tion of 68Ga-DOTA-4-FBn-TN14003 both Jurkat and MDA-MB-231
cells were used. The radiotracer was added in a concentration of
250 nM and the cells were prepared as described above. For tracer
uptake kinetics the cells were incubated at 37 °C for 5, 10, 15, 20,
30, 60, 90, and 120 min, respectively, and the probe workup was
conducted as described for the competition binding assays. Exper-
iments were performed 3 times including quadruplicate sample
measurement and binding results were expressed as % cell bound
activity to total activity.
For the internalization assays cell lines were incubated for 1 h at
37 °C. After incubation, cells were centrifuged for 2 min at
2000 rpm and the supernatant was removed. Subsequently, cells
were washed 3 times with cold PBS and then, for removal of sur-
face-bound radioactivity, 2 times for 5 min with 50 mM Glycine
buffer (pH 2.8) at 4 °C. After washing with cold PBS once, the cell
pellet was resuspended in 0.5 M NaOH. The radioactivity of the
2.6. Cell lines and cell culture
The human T lymphocyte cell line Jurkat was purchased from
European Collection of Cell Cultures (ECACC). Jurkat cells were cul-
tured routinely in RPMI-1640 (Gibco, Invitrogen, Karlsruhe, Ger-
many), supplemented with 10% FCS (Sigma, Taufkirchen,
Germany). All cultures were kept under controlled conditions
(humidified atmosphere, 5% CO2, 37 °C) and passaged 2–3 times
a week to keep them in logarithmic growth. Sub-confluent Jurkat
cells were harvested, counted on a Neubauer’s chamber and resus-
pended in binding buffer18 (Dulbecco’s PBS containing 20 mM
HEPES and 0.5% BSA, pH 7.0) to a concentration of 1.1 ꢀ 107 in
400 lL. The human breast cancer cell line MDA-MB-231 was pur-
chased from American Type Culture Collection (ATCC). MDA-MB-
231 cells were cultured routinely in RPMI-1640, supplemented
with 10% FCS and 1% penicillin/streptomycin (Gibco, Invitrogen,
Karlsruhe, Germany). All cultures were kept under controlled con-
ditions (humidified atmosphere, 5% CO2, 37 °C) and passaged 2–3
times a week to keep them in logarithmic growth. Sub-confluent
MDA-MB-231 cells were harvested using 0.05% Trypsin–EDTA
(Gibco, Invitrogen, Karlsruhe, Germany) and prepared as described
above.
samples was measured in a
c-counter. Experiments were per-
formed 3 times including triplicate sample measurements. The val-
ues are expressed as the percentage of internalized radioactivity
(cell pellet) to total activity (glycine washes, last PBS wash and cell
bound activity).
2.9. Immunocytochemical staining
The cytochemical staining for CXCR4 was performed with Jurkat
and MDA-MB-231 cells in a concentration of 1 ꢀ 106 cells per mil-
liliter, respectively. As primary antibody human CXCR4 (monoclo-
nal mouse IgG2A, clone 12G5; dilution 1:100) from R&D Systems
(Minneapolis, USA) and as secondary antibody Texas-Red-conju-
gated goat anti-mouse (dilution 1:100; Dianova, Hamburg, Ger-
many) were used. Counterstaining of cell nuclei was conducted
with DAPI (40,6-diamidino-2-phenylindole, dilution 1:500, Invitro-
gen, Karlsruhe, Germany). For staining the cells were either fixated
on glass plates (for images) or unfixated in PBS (for analysis). With
the primary antibody cells were incubated for 60 min and with the
secondary antibody together with DAPI for 30–45 min at room
temperature. The stainings were evaluated using a fluorescence
microscope BX50 with an adapted digital camera (F-View) from
Olympus Soft Imaging Solutions (Muenster, Germany). Positive
area fractions (10 fields of view per staining) were analyzed with
cellF software (Olympus Soft Imaging Solutions, Muenster, Ger-
many) using unfixated cells (magnification 20-fold, exposure time
1 s).
2.7. Cell binding assays using 125I-SDF-1
a
In order to determine the binding affinity of Ga-DOTA-4-FBn-
TN14003 and 4-FBn-TN14003 (for comparison) to CXCR4 on living
cells, a competitive cell binding assay was performed using Jurkat
as well as MDA-MB-231 cells. The assay was modified from a pub-
lished procedure.18 For the binding assay with Jurkat cells, the cells
were harvested and resuspended in binding buffer (Dulbecco’s PBS
containing 20 mM HEPES and 0.5% BSA, pH 7.0). A suspension of
105 cells, 0.055 nM human 125I-SDF-1
a (specific activity: 2200 Ci/
mmol, Perkin–Elmer Life Sciences, Boston, MA, USA) and 12 differ-
ent concentrations of Ga-DOTA-4-FBn-TN14003 (0–5000 nM) were
incubated for 1 h at 37 °C. It was essential that for the binding reac-
tion as well as the previous dilution step Protein LoBind vials
(Eppendorf) were used because otherwise 125I-SDF-1
a could not
be recovered sufficiently. The incubation was terminated by
removing aliquots of the mixture and separating cells from buffer
by centrifugation (12,000 rpm) through a silicone/mineral oil mix-
ture. The cell-bound radioactivity as well as the non-bound radio-
3. Results and discussion
activity was measured using a
c-counter. Experiments were
performed at least 4 times including quadruplicate sample mea-
surements. Binding results were expressed as % cell bound activity
to total activity and the IC50 values were calculated using Sigma
The peptide 4-FBn-TN14003 (1, Fig. 1a) was first described by
Tamamura et al. as an antagonist for CXCR4 and was developed

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U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
by improving the structure of T140 analogs.18 It was shown that
this peptide has nanomolar affinity for the chemokine receptor,
that it is stable in vivo and that it reduces breast cancer lung
metastasis in SCID mice. Like the parent T140 this peptide consists
of 14 amino acid residues and differs from T140 by the C-terminal
amide as well as the N-terminal 4-fluorobenzoyl group. These
changes resulted in an improved bio-stability and affinity for
CXCR4.18 In order to make this peptide available as a PET or SPECT
tracer, different radiolabeling approaches are feasible. One possi-
bility is the direct electrophilic radioiodination of tyrosine residues
in 4-FBn-TN14003 which has already been done successfully
(unpublished results). However, this approach has several disad-
vantages and has therefore not longer been pursued. As two differ-
ent tyrosine residues are present in the amino acid sequence of the
peptide (Tyr5 and Tyr10), both residues could be radioiodinated.
Therefore two different labeled compounds would be formed
which were expected not to be easily separated chromatographi-
cally and to also exhibit different pharmacokinetics. Another cause
of concern is that Tyr5 is part of the intrinsic pharmacophore (red
amino acid residues in Fig. 1) necessary for binding to CXCR419–21
and a radioiodination might potentially lead to a decreased binding
efficiency. Another labeling approach represents the 18F-fluorina-
tion of TN14003 using 18F-SFB as a secondary labeling precursor
which has been reported recently.22 18F-4-FBn-TN14003 was eval-
uated in mice bearing CHO-CXCR4 tumors in biodistribution as
well as PET studies and showed promising results. However, this
labeling approach has also disadvantages which will be discussed
below. A completely different radiolabeling approach is the site-
benzoic acid followed by the formation of the disulfide bridge be-
tween Cys4 and Cys13. For this purpose the Acm-groups were
selectively deprotected on the resin using thallium(III) trifluoro-
acetate and the disulfide bridge was formed.25 The formation of
the disulfide bridge was performed before DOTA conjugation to
D
-Lys8 because otherwise disulfide ring formation might be im-
peded due to steric hindrance. Selective DOTA conjugation was
achieved at -Lys8 using Mtt protection which was easily cleaved
D
with 1.5% TFA while Lys7 was protected by a Boc group which is
stable under these mild conditions. The tris-tBu-DOTA derivative
needed for conjugation to the peptide was synthesized via a three
step reaction procedure starting from commercially available cyc-
lene [similar to15]. Tris-tBu-DOTA was activated by HBTU and cou-
pled to D-Lys8. The final cleavage and deprotection yielded DOTA-
4-FBn-TN14003. The reference peptide 4-FBn-TN14003 was syn-
thesized accordingly omitting the conjugation of tris-tBu-DOTA.
This synthesis strategy allowed the execution of all reaction steps
on the solid phase support including the site-specific conjugation
of DOTA to D-Lys8 of 4-FBn-TN14003 and the disulfide bridge for-
mation. With DOTA-4-FBn-TN14003 a versatile labeling precursor
for (radio)metal complexation was obtained. Depending on the
radiometal used for complexation, the compound enables the syn-
thesis of either a PET or SPECT tracer for the imaging of CXCR4 in
tumors and metastases or a radiopharmaceutical for use as a ther-
apeutic agent.
3.2. 68Ga-Labeling of DOTA-4-FBn-TN14003
specific conjugation of the peptide 4-FBn-TN14003 at the
e
-amino
In order to synthesize a PET tracer for the imaging of CXCR4
expression in tumors and metastases, the CXCR4 inhibitor DOTA-
4-FBn-TN14003 was labeled with 68Ga. The radioactive labeling
was optimized concerning various reaction parameters such as
the amount of labeling precursor, pH value, reaction temperature,
and reaction time using analytical reversed-phase HPLC. The radio-
labeled product eluted in a single peak with a retention time of
3.31 min and its identity was confirmed by co-elution with the
non-radioactive reference compound Ga-DOTA-4FBn-TN14003.
Concerning the amount of labeling precursor, no significant differ-
ence in RCY could be observed when using 5, 10 or 15 nmol of the
precursor peptide. Depending on the application of the radiotracer,
the labeling reactions were therefore performed with either 5 or
10 nmol precursor. Varying the pH value of the reaction solution
between 4 and 5 did not result in significant differences in RCY
as well and the same was observed for the reaction time (8 and
18 min, respectively) and the reaction temperature (80 and 90 °C,
respectively). For subsequent experiments a reaction temperature
of 80 °C and a reaction time of 10 min were chosen because a
further increase in reaction time would only lead to a loss of
radioactivity due to the radioactive decay of 68Ga. Routinely RCYs
of 95–100% (n >15) were achieved determined by analytical HPLC.
For the evaluation of 68Ga-DOTA-4-FBn-TN14003 in cell assays,
no further purification of the compound was necessary. When nec-
essary, however, purification of the radiotracer was accomplished
by solid phase extraction (SPE) using a C18 cartridge. The radio-
chemical purity of purified 68Ga-DOTA-4-FBn-TN14003 was deter-
mined by HPLC to be >99.5% and the isolated RCY was 72.5 4.9%.
At the end of synthesis specific activities of 68Ga-DOTA-4-FBn-
function of a lysine residue with a suitable chelator for the com-
plexation of radiometals. Appropriate chelators are for example
DTPA (diethylenetriaminepentaacetic acid; for labeling with the
SPECT isotope 111In) or DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid; for labeling with either 111In, the PET iso-
topes 64Cu and 68Ga or the therapy isotopes 90Y and 177Lu). A sim-
ilar ligand for CXCR4, namely Ac-TZ14011, has already been
conjugated with DTPA and labeled with 111In showing promising
results in first evaluation studies.10 In this peptide the Lys7 residue
of T140 was changed to an Arg residue in order to achieve site-
selective conjugation of the chelator moiety to
conjugation did not interfere with the binding efficiency of the
peptide to CXCR4 because the -Lys8 residue is not part of the
pharmacophoric structure. Another recently published approach
D-Lys8. The DTPA
D
was the conjugation of two DOTA moieties to both Lys7 and D-
Lys8 and labeling of the peptide with PET radiometals.23 Since we
are also interested in a radiometallated PET and not a SPECT tracer
for CXCR4, 68Ga was the isotope of our choice. 68Ga combines well-
suited decay properties as a PET isotope with its easy availability
via a generator.24 A suitable chelator for 68Ga is DOTA which was
site-selectively conjugated to the
D-Lys8 residue of 4-FBn-
TN14003 (2, Fig. 1b). Without the need to exchange Lys7 and still
obtaining one specific product and one specific side for labeling,
we received this peptide for radiolabeling with 68Ga and other
potentially suited radiometals such as 90Y or 177Lu.
3.1. Synthesis of DOTA-4-FBn-TN14003 and 4-FBn-TN14003
DOTA-4-FBn-TN14003 as well as 4-FBn-TN14003 were received
by Fmoc-based solid phase synthesis on Rink Amide resin using
amino acid derivatives with suitable protection groups, for exam-
TN14003 of up to 29.8 2.3 GBq/lmol were achieved depending
on the amount of labeling precursor as well as the starting radioac-
tivity and the RCY of the labeling reaction. The overall time for syn-
thesis and isolation of 68Ga-DOTA-4-FBn-TN14003 was 35 min,
which is compatible with the half-life of 68Ga (68 min). Due to
the simplicity of the labeling and purification procedures, the
labeling protocol may also be adoptable for automation.
ple, the highly acid-labile 4-methyltrityl (Mtt) group for
D-Lys
and the acetamidomethyl (Acm) group for Cys. The synthesis route
is shown in Figure 2. All reaction steps, including DOTA conjuga-
tion and formation of the disulfide bond, could be conducted on
the solid phase support. After stepwise condensation of the 14
amino acid residues, the N-terminus was acylated with 4-fluoro-
As mentioned above, the CXCR4 inhibitor 4-FBn-TN14003 has
already been described as a 18F-labeled tracer. However, the

U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
1507
following advantages of labeling a peptide with 68Ga as compared
to 18F exist: first, the availability of 68Ga via a 68Ge/68Ga-generator
whereas for the production of 18F a cyclotron is needed. Secondly,
for 18F-fluorinations a larger amount of labeling precursor is
needed which requires a HPLC separation step, removal of HPLC
solvent and formulation of the final product. Thirdly, the 18F-fluo-
rination of a peptide usually necessitates a secondary labeling
precursor such as 18F-SFB.26,27 This secondary labeling precursor
was also used for the preparation of 18F-4-FBn-TN14003 and
resulted in a 5 step reaction procedure, 3 steps for the synthesis
of 18F-SFB and another 2 steps for the conjugation to the peptide
precursor.22 The elaborate labeling procedure resulted in an overall
reaction time of more than 165 min. As long as the derivatization
of peptides with a chelator and the 68Ga complexation does not
lead to a considerable decrease in affinity the radiometal complex-
ation is preferable.
well as 4-FBn-TN14003. The competition curves for both com-
pounds are depicted in Figure 3, showing that the plateau phases
for low as well as high concentrations of the peptides are reached,
respectively. Table 1 summarizes the IC50 values (inhibitory con-
centrations of 50%) for both compounds and both cell lines. For
4-FBn-TN14003 the IC50 value was determined to be
4.07 1.00 nM (n = 5) indicating a high affinity of the peptide to
CXCR4. This result was in the same range as previously published
values for this peptide (2.5 nM using CHO-CXCR4 cells22 and
0.99 nM using Jurkat cells18). The IC50 value of Ga-DOTA-4-FBn-
TN14003 was 1.99 0.31 nM (n = 4) under the same assay
conditions demonstrating similar CXCR4 affinity. Obviously, the
Ga-DOTA substituent had no influence on the affinity of the origi-
nal 4-FBn-TN14003. This result was expected because for a similar
inhibitor for CXCR4, namely Ac-TZ14011, the affinity of In-DTPA-
Ac-TZ14011 was only slightly lower than that of the parent peptide
(IC50 = 7.9 nM vs 1.2 nM, respectively10).
3.3. Stability of 68Ga-DOTA-4-FBn-TN14003
When using MDA-MB-231 cells instead of Jurkat cells down-
scaled competition curves were observed (Fig. 4), because the max-
The stability of 68Ga-DOTA-4-FBn-TN14003 in PBS and human
plasma was evaluated at 37 °C. The radiochemical stability of the
compound in PBS was examined by repeated HPLC analysis, prov-
ing stability over a period of at least 4 h. In human plasma, the sta-
bility was evaluated using HPLC as well as size exclusion
chromatography (SEC) after 30 and 90 min incubation time. After
30 min the radiochemical purity of the radiotracer was >99% and
after 90 min >97% indicating 3% free 68Ga. The recovery of the
radioactive sample from the HPLC was 100%. Using SEC the binding
of the radiotracer to plasma can be evaluated. After 90 min >90% of
the injected sample corresponded to non-bound radiotracer and
the recovery from SEC was also 100%. Taken together, these results
suggest a high stability of 68Ga-DOTA-4-FBn-TN14003 which is an
important property of a radiotracer.
imal cell binding of 125I-SDF-1
a was significantly lower. Looking at
the parent peptide 4-FBn-TN14003, the maximal binding was re-
duced from 19.8 1.0% (Jurkat, n = 5) to 10.4 0.3% (MDA-MB-
231, n = 4), respectively. Accordingly, for Ga-DOTA-4-FBn-
TN14003 the maximal binding was reduced from 18.5 0.8% (Jur-
kat, n = 4) to 8.4 0.3% (MDA-MB-231, n = 5). These results suggest
that MDA-MB-231 human breast cancer cells express less CXCR4
than human T lymphocyte Jurkat cells. To verify this observation,
the expression of CXCR4 receptors on Jurkat and MDA-MB-231
cells was studied using immunocytochemical staining as well as
a competition binding assay (see Section 3.4.3).
Besides the lower maximal binding, another difference between
MDA-MB-231 and Jurkat cells was observed in the lower IC50 val-
ues for both peptides (Table 1). Due to the higher error bars as well
as the downscaled competition curves the fits of the respective
curves for the determination of the IC50 values were not as good
as for the Jurkat cell experiments which might explain the discrep-
ancy between the IC50 values for both cell lines. Even though the
expression of CXCR4 on MDA-MB-231 cells is lower, the higher
affinity of both peptides might on the other hand be caused by a
slight variance of the receptor on this cell line. The conformation
of the receptor might be better suited to the peptides. Slight differ-
ences in the affinity of 4-FBn-TN14003 to CXCR4 when using dif-
ferent cell lines were also described in previously published
evaluations (see above).18,22 To verify these considerations, further
experiments should be performed using a panel of cell lines with
different CXCR4 expression levels.
3.4. In vitro evaluation
3.4.1. Determination of the binding affinity to CXCR4
An essential property for the suitability of a compound as a PET
or SPECT tracer is its affinity to the receptor of choice. While it is
known that 4-FBn-TN14003 has a high affinity for CXCR4, conjuga-
tion of a chelator like DOTA including complexation with gallium
might change the receptor affinity significantly. For the determina-
tion of the affinity of Ga-DOTA-4-FBn-TN14003 to CXCR4 the non-
radioactive complex was synthesized and a competitive cell bind-
ing assay was performed using 125I-SDF-1
a, the natural ligand for
CXCR4, as the specific radioligand. To evaluate the influence of
the Ga-DOTA complex, the affinity of the parent peptide 4-FBn-
TN14003 was determined accordingly. For this purpose Jurkat cells
(derived from a human T cell lymphoma), which are known to
highly express CXCR4 receptors,28 were used as well as MDA-
MB-231 human breast cancer cells which also express the recep-
tor.18 For the evaluations vital cells were used which provide real-
istic conditions of receptor expression on the cell surface. Initially
some difficulties were encountered concerning the recovery of
The competitive binding assays for both cell lines showed the
high affinity of the parent peptide 4-FBn-TN14003 as well as the
novel compound Ga-DOTA-4-FBn-TN14003. It could be demon-
strated that the modification of the parent peptide did not result
in a decrease of the affinity to CXCR4 making this peptide a prom-
ising candidate as a new PET tracer.
3.4.2. Uptake kinetics and internalization properties of 68Ga-
DOTA-4-FBn-TN14003
125I-SDF-1
a
a
during the assay. Because of the strong adherence of
to safe lock plastic vials (Eppendorf) when using cell
125I-SDF-1
For estimating the optimal imaging time point in regard to
in vivo studies using 68Ga-DOTA-4-FBn-TN14003, the uptake
kinetics of the tracer were evaluated in Jurkat as well as MDA-
MB-231 cells. Fast uptake of the ligand was observed in both cell
lines (Fig. 5), and again lower uptake for MDA-MB-231 cells could
be observed as compared to Jurkat cells. While the uptake in MDA-
MB-231 cells remained stable over time, at 90 and 120 min a de-
crease in uptake was observed for Jurkat cells. These results sug-
gest that the kinetics of 68Ga-DOTA-4-FBn-TN14003 are fast,
which is expected for a small peptide, and that the half-life of
68Ga is suitable for PET imaging using this radiotracer.
medium supplemented with 10% FCS as assay buffer, different con-
ditions were evaluated. The use of screw cap vials as well as the
use of cell medium supplemented either with 0.25% BSA or without
serum did not improve the results. It was essential to perform the
assay in protein LoBind vials (Eppendorf) which are special vials
with reduced binding of proteins to the surface. With these vials
recoveries of 97.9 1.9% of the radioligand 125I-SDF-1
achieved.
a were
First, the cell binding assays were performed using Jurkat cells
and evaluating the affinity of both Ga-DOTA-4-FBn-TN14003 as

1508
U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
(a)
(b)
20
15
10
5
20
15
10
5
0
0
-4
-2
0
2
4
-4
-2
0
2
4
log c(4-FBn-TN14003)
log c(Ga-DOTA-4-FBn-TN14003)
Figure 3. Competitive binding assay of 4-FBn-TN14003 (a) and Ga-DOTA-4-FBn-TN14003 (b), respectively, against 125I-SDF-1
a
on Jurkat cells. Concentrations of the
inhibitors were varied between 0–5000 nM. Values are mean standard deviation (n = 5 (a) and 4 (b), including quadruplicate sample measurements).
considerable difference in the maximal binding to both cell lines
could be observed. These results suggest that MDA-MB-231 breast
Table 1
IC50 values of 4-FBn-TN14003 and Ga-DOTA-4-FBn-TN14003 using Jurkat as well as
MDA-MB-231 cells in a competitive binding assay with 125I-SDF-1
a
cancer cells express significantly less CXCR4 receptors than Jurkat
T lymphocyte cells. For verification, the expression levels on both
cell lines were determined by immunocytochemical staining for
CXCR4 as well as a competition radioligand binding assay. The
immunocytochemical staining for CXCR4 is shown in Figure 6 to-
gether with an analysis of the mean positive area fractions. In com-
parison, Jurkat cells expressed high amounts of CXCR4, whereas
MDA-MB-231 cells expressed considerably less CXCR4.
IC50 (nM)
Jurkat
MDA-MB 231
4-FBn-TN14003
Ga-DOTA-4-FBn-TN14003
4.07 1.00
1.99 0.31
1.75 0.54
1.04 0.33
Values are mean standard deviation (n = 4 or 5).
Using a competitive binding assay by inhibiting the binding of
human 125I-SDF-1
a by various concentrations of human SDF-1a,
In order to determine how much of the radiotracer that bound
to the cells was actually internalized, the cells were washed with
an acidic buffer to cleave off surface-bound radioactivity. At
37 °C, the internalization was 29.5 1.8% for Jurkat cells and
40.0 0.5% for MDA-MB-231 cells, respectively. These data are in
line with recently published results for a similar peptide (64Cu-
T140-2D), which has two DOTA moieties conjugated to both lysine
residues in the 14 amino acid peptide 4-FBn-TN14003.23 Using this
peptide, internalization of one third to one half of the radioactivity
was observed in CHO-CXCR4 cells at 37 °C.
the determination of Bmax, the number of binding sites per cell,
was possible. In comparison to Jurkat cells, which expressed
(3.40 0.64) ꢀ 105 receptors per cell, MDA-MB-231 cells showed
only (1.13 0.44) ꢀ 104 sites per cell. This explains the results ob-
tained above. As reported previously, Jurkat cells express CXCR4 on
a high level,28 which could be verified here in both assays. For
MDA-MB-231 cells the expression of CXCR4 was also reported by
Tamamura et al. as verified by RT-PCR.18 In a recent publication
by Nimmagadda et al. MDA-MB-231 cells were characterized as
low expressing CXCR4 cells by flow cytometric analysis.13 In a tu-
mor model of MDA-MB-231-derived lung metastases in mice the
same authors showed that the visualization of these metastases
with 64Cu-AMD3100 was feasible and that the expression of CXCR4
3.4.3. Expression of CXCR4
During the binding assays with 125I-SDF-1 and 68Ga-DOTA-4-
a
FBn-TN14003 using Jurkat and MDA-MB-231 cells, respectively, a
12
12
(a)
(b)
10
8
10
8
6
6
4
4
2
2
0
0
-4
-2
0
2
4
-4
-2
0
2
4
log c(4-FBn-TN14003)
log c(Ga-DOTA-4-FBn-TN14003)
Figure 4. Competitive binding assay of 4-FBn-TN14003 (a) and Ga-DOTA-4-FBn-TN14003 (b), respectively, against 125I-SDF-1
a
on MDA-MB-231 cells. Concentrations of the
inhibitors were varied between 0–5000 nM. Values are mean standard deviation (n = 4 (a) and 5 (b), including quadruplicate sample measurements).

U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
1509
(a)
(b)
10
10
8
8
6
4
2
0
6
4
2
0
0
20
40
60
80
100
120
0
20
40
60
80
100
120
t / min
t / min
Figure 5. Uptake kinetics of 68Ga-DOTA-4-FBn-TN14003 on Jurkat (a) and MDA-MB-231 cells (b), respectively. Values are mean standard deviation (n = 3, including
quadruplicate sample measurements).
Figure 6. Immunocytochemical analysis of CXCR4: (a) Jurkat and (b) MDA-MB-231. Colored images show merged signal channels for CXCR4 (red) and DAPI (blue).
Magnification 40-fold. (d) Mean positive area fractions for CXCR4 staining. Values are mean standard deviation (n = 10).
in the metastatic tissue was increased by 15–30%. These results
indicate that the visualization of metastases by specific imaging
of CXCR4 should be possible with 68Ga-DOTA-4-FBn-TN14003,
which we introduced here, due to its significantly higher affinity
for CXCR4 than the bis(cyclam) complex 64Cu-AMD3100
planned to be further evaluated in suited animal models of
metastases.30
4. Conclusions
(IC50 = 1.99 0.31 nM vs 62.7
l
M,29 respectively) and due to the
The favorable binding characteristics of 68Ga-DOTA-4-FBn-
TN14003 to CXCR4, as well as its site-specific solid phase peptide
synthesis and easily adoptable radiosynthesis, gives rise to the sig-
nificant potential of this compound as a novel PET tracer. Future
favorable properties of 68Ga as a PET isotope in comparison to
64Cu (b+ decay is only 17.6%24). The potency of 68Ga-DOTA-4-
FBn-TN14003 for the imaging of disseminated tumor cells is

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U. Hennrich et al. / Bioorg. Med. Chem. 20 (2012) 1502–1510
10. Hanaoka, H.; Mukai, T.; Tamamura, H.; Mori, T.; Ishino, S.; Ogawa, K.; Iida, Y.;
Doi, R.; Fujii, N.; Saji, H. Nucl. Med. Biol. 2006, 33, 489.
11. Masuda, R.; Oishi, S.; Ohno, H.; Kimura, H.; Saji, H.; Fujii, N. Bioorg. Med. Chem.
2011, 19, 3216.
experiments are currently underway to evaluate its usefulness for
the detection of tumors and especially metastases.
12. Nimmagadda, S.; Pullambhatla, M.; Pomper, M. G. J. Nucl. Med. 2009, 50, 1124.
13. Nimmagadda, S.; Pullambhatla, M.; Stone, K.; Green, G.; Bhujwalla, Z. M.;
Pomper, M. G. Cancer Res. 2010, 70, 3935.
14. De Silva, R. A.; Peyre, K.; Pullambhatla, M.; Fox, J. J.; Pomper, M. G.;
Nimmagadda, S. J. Nucl. Med. 2011, 52, 986.
15. Wängler, C.; Wängler, B.; Eisenhut, M.; Haberkorn, U.; Mier, W. Bioorg. Med.
Chem. 2008, 16, 2006.
16. Mühlhausen, U.; Ermert, J.; Coenen, H. H. J. Labelled Compd. Radiopharm. 2009,
52, 13.
Acknowledgments
The authors wish to thank Dr. William E. Hull (core facility
Molecular Structure Analysis) for the characterization of DOTA-4-
FBn-TN14003 by NMR. The authors wish to thank the Deutsche
Forschungsgemeinschaft (BA 4027/4-1) for their financial support
(U.H. and T.B.).
17. Schuhmacher, J.; Maier-Borst, W. Int. J. Appl. Rad. Isot. 1981, 32, 31.
18. Tamamura, H.; Hori, A.; Kanzaki, N.; Hiramatsu, K.; Mizumoto, M.; Nakashima,
H.; Yamamoto, N.; Otaka, A.; Fujii, N. FEBS Lett. 2003, 550, 79.
19. Tamamura, H.; Omagari, A.; Oishi, S.; Kanamoto, T.; Yamamoto, N.; Peiper, S. C.,
et al Bioorg. Med. Chem. Lett. 2000, 10, 2633.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.052.
20. Tamamura, H.; Sugioka, M.; Odagaki, Y.; Omagari, A.; Kan, Y.; Oishi, S., et al
Bioorg. Med. Chem. Lett. 2001, 11, 359.
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22. Jacobson, O.; Weiss, I. D.; Kiesewetter, D. O.; Farber, J. M.; Chen, X. J. Nucl. Med.
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