Magnetic resonance imaging of tumor cells by targeting the amino acid transport system

Article abstract of DOI:10.1016/j.bmcl.2006.04.081

An early diagnosis of cancer is crucial in the battle against this disease and the in vivo visualization of tumors at cellular level is still the most challenging goal. In order to target tumor cells, we took into account their increased metabolism and amino acid nutrients or pseudo-nutrients, which are actively transported through the cell membrane, have been chosen as vectors for new MRI contrast agents. For this reason new gadolinium complexes conjugated to agmatine, arginine, and glutamine have been synthesized and studied.

Full text of DOI:10.1016/j.bmcl.2006.04.081

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4111–4114
Magnetic resonance imaging of tumor cells by targeting
the amino acid transport system
Luciano Lattuada,a,* Silvia Demattio,a Veronica Vincenzi,a Claudia Cabella,b
Massimo Visigalli,b Silvio Aime,c Simonetta Geninatti Crichc and Eliana Gianolioc
aCRM Chemistry, Bracco Imaging SpA, via E. Folli 50, 20134 Milano, Italy
bCRM Chemistry, Bracco Imaging SpA, via Ribes 5, 10010 Colleretto Giacosa (TO), Italy
c
`
Dipartimento di Chimica IFM, Universita di Torino, via P. Giuria 7, 10125 Torino, Italy
Received 15 March 2006; revised 26 April 2006; accepted 26 April 2006
Available online 18 May 2006
Abstract—An early diagnosis of cancer is crucial in the battle against this disease and the in vivo visualization of tumors at cellular
level is still the most challenging goal. In order to target tumor cells, we took into account their increased metabolism and amino
acid nutrients or pseudo-nutrients, which are actively transported through the cell membrane, have been chosen as vectors for new
MRI contrast agents. For this reason new gadolinium complexes conjugated to agmatine, arginine, and glutamine have been
synthesized and studied.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Magnetic resonance imaging (MRI) is a powerful, non-
invasive, and widely applied diagnostic technique which
allows to obtain images of the inside of the human
body.1,2 Nowadays, more than one-third of the MRI
scans are performed by administration of a contrast
agent, usually a gadolinium complex.3,4 Gadolinium
(III) ion is able, due to its favorable paramagnetic prop-
erties, to increase the relaxation rate of the surrounding
water protons, making the region of interest brighter
than the background.
cells, allowing an accurate diagnosis of the disease when
it is still treatable.
A well-assessed method of tumor diagnosis is the target-
ing of over-expressed receptors on the membrane of
tumor cells with specific radiopharmaceuticals,8–11 but
in spite of its high sensitivity this technique only gives
poor image resolution. In the case of MRI this targeting
strategy is hampered by the very low concentration of
such receptors12 and by the poor sensitivity of gadolini-
um complexes. Among the several possibilities proposed
to achieve an efficient cellular uptake of gadolinium
complexes13 we took into account transport systems,
which are more efficient in terms of internalization than
receptors. Considering that rapidly growing tumors
require an increased and continuous supply of both
essential and nonessential amino acids, we selected the
amino acid transport system. In particular, we focused
on glutamine transport system because glutamine is
the main source of nitrogen for tumor cells.14,15 For
these reasons DTPA (diethylenetriaminepentaacetic
acid) and DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid) were conjugated to glutamine,
arginine, and agmatine to give six new gadolinium com-
plexes that were studied in terms of relaxivity, tumor
internalization, and in vitro MRI imaging.
Since the approval of the first gadolinium complex for
human administration in 1988, several other analogues
have reached the market.5,6 The contrast agents of first
generation distribute into the intravascular and intersti-
tial space immediately after injection and in this context
are called ‘‘non-specific agent’’. The medical need for
tissue specific contrast agents has driven researchers to
design and synthesize contrast agents of second genera-
tion able to visualize selectively the liver or the cardio-
vascular system, for instance.7 The ultimate goal is a
contrast agent that accumulates specifically in tumor
Keywords: Magnetic resonance imaging; Gadolinium complexes;
Glutamine uptake; Tumor cells; Amino acid transporters.
For the synthesis of DTPA derivatives, DTPA-Glu 116
was reacted with N-hydroxysuccinimide and N-ethyl-
*
Corresponding author. Tel.: +39 02 21772621; fax: +39 02
21772794; e-mail: luciano.lattuada@bracco.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.081

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L. Lattuada et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4111–4114
N0-(3-dimethylaminopropyl)carbodiimide (EDCI) in
CH2Cl2 to give the N-hydroxysuccinimidyl ester 5 which
was directly coupled without purification to glutamine
tert-butyl ester 2, bis-Boc-agmatine 3,17 and argi-
nine(Mtr) tert-butyl ester 4.
tBuOOC
COOtBu
ROC
COOH
COOH
N
N
N
N
N
N
N
N
a,b
tBuOOC
COOH
HOOC
9a,b
8
COO-
The corresponding amides obtained were deprotected
with trifluoroacetic acid (TFA) to give acyclic ligands
6a–c. Complexation was performed by heating the sus-
pension of ligand and Gd2O3 in water at 50 ꢁC for
16 h. The excess of Gd2O3 was filtered off, 1 M NaOH
was added until pH 7, and the solution evaporated to
give complexes 7a–c as sodium salts (Scheme 1).
ROC
N
N
N
c
Gd3+
N
-OOC
COO-
10a,b
NH
H NOC
R =
2
COOH
a
b
R =
H2N
NH
NH
NH
For the synthesis of DOTA derivatives, DOTA tris tert-
butyl ester 818 was coupled to amines 2 and 3 using
EDCI, 1-hydroxybenzotriazole (HOBt), and diethyliso-
propylamine (DIEA) in CH2Cl2. The corresponding
amides obtained were deprotected with TFA and the
cyclic ligands 9a,b so obtained were reacted with
Gd2O3 to give gadolinium complexes 10a,b (Scheme 2).
Scheme 2. Reagents and conditions: (a) amine 2 or 3, HOBt, EDCI,
DIEA; (b) TFA, (c) Gd2O3, H2O, 50 ꢁC.
(4.3 sꢀ1 mMꢀ1
)
and Gd-DOTA (4.2 sꢀ1 mMꢀ1
)
19
19
complexes. Since the increase in relaxivity is higher than
that expected from the effects associated to the elonga-
tion of the molecular reorientational time, a possible
explanation may be ascribed to a contribution arising
from water molecules/exchangeable protons in the sec-
ond coordination sphere due to the presence of the ami-
noacid residue.
Complex 10c was obtained with a different synthetic
strategy. Arginine(Mtr) tert-butyl ester 4 was acylated
with chloroacetyl chloride to give chloroacetamide 11.
DO3A tris tert-butyl ester 1218 was thus alkylated with
11 and the product obtained deprotected with TFA to
give cyclic ligand 9c. Final complexation with Gd2O3
gave product 10c (Scheme 3).
Cellular uptake (Fig. 1) was performed with rat hepato-
carcinoma cell line (HTC).20 The cells were seeded in
75 cm2 flasks at a density of about 25,000 cells/cm2.
After 24 h, cells were washed with 5 mL phosphate-
buffered saline (PBS) and then incubated at 37 ꢁC for
6 h with 5 mL of Earle’s balanced salt solution (EBSS)
in the presence of 1.6 mM concentration of each Gd(III)
complex. At the end of the uptake/binding experiment
the medium was removed; the cells (ca. 5 · 106) were
washed three times with 5 mL ice-cold PBS and collect-
ed in 200 lL PBS. Cells were then treated with 200 lL of
Deprotection of DTPA-Glu 1 with TFA and complexa-
tion with Gd2O3 gave the reference compound 13
(Scheme 1).
Relaxivity (r1) of all gadolinium complexes (Table 1)
was determined by titration of an aqueous solution of
each corresponding ligand with a standard solution
of GdCl3. All complexes show relaxivities significantly
higher than the ones of the parent Gd-DTPA
O
N
HOOC
tBuOOC
tBuOOC
COOtBu
N
OOC
COOtBu
N
ROC
N
COOH
O
a
b,c
N
N
N
N
COOtBu
COOtBu
tBuOOC
N
COOtBu
COOtBu
HOOC
N
COOH
COOH
1
5
tBuOOC
HOOC
6a-c
c,d
d
-OOC
COO-
COO-
ROC
N
N
N
-OOC
-OOC
COO-
COO-
N
N
-OOC
COO-
COO-
N
Gd3+ 3Na+
Gd3+
-OOC
7a-c
13
NH
H NOC
2
COOH
NH
NH
a R =
b R =
H2N
c R =
H2N NH
NH
COOH
NH
NH
Scheme 1. Reagents and conditions: (a) N-hydroxysuccinimide, EDCI, CH2Cl2; (b) coupling with amines 2–4; (c) TFA; (d) Gd2O3, 1 M NaOH, pH
7, 50 ꢁC.

L. Lattuada et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4111–4114
4113
Me
Me
NH
NH
Gd-DOTA-Gln 10a
COOtBu
NH2
Gd-DOTA-Agm 10b
Gd-DOTA-Arg 10c
Gd-DTPAGlu-Gln 7a
Gd-DTPAGlu-Agm 7b
Gd-DTPAGlu-Arg 7c
Gd-DTPA-Glu 13
MeO
SO2NH
4
Me
a
Me
Me
NH
COOtBu
MeO
SO2NH
NH
NHOC
Cl
Me
11
b,c
0
0,5
1
1,5
2
2,5
3
3,5
4
Gd moles/mg protein x108
COOH
NHOC
H2N
NH
NH2
Figure 1. Comparison of cellular uptakes of the gadolinium complexes
synthesized in this work on hepatocytes (as model of healthy cells) and
HTC (as model of tumor cells). Each value is the average of three
experiments.
COOH
COOH
N
N
N
N
HOOC
d
9c
For MRI of cell pellets (Fig. 2), cells were incubated with
the contrast agent, extensively washed, detached, and
again washed as described above. The cells were pelleted
within a glass capillary (1 mm diameter) that was then
inserted in 2% agarose within a 5 ml tube. The imaging
protocol consisted of an Inversion-Recovery T1-weighted
spin-echo.21 The T1 of the hepatocytes blank is shorter
than HTC blank because of their different iron content.
The membrane binding (4 ꢁC) of compound 7a is higher
in HTC cells because of the over-expression of the amino
acid transport systems on these tumor cells.22
COO-
H2N
NH
COO-
COO-
NHOC
+
N
N
N
NH2
Gd3+
N
-OOC
10c
Scheme 3. Reagents and conditions: (a) chloroacetylchloride, DIEA,
CH2Cl2; (b) DO3A tris tert-butyl ester 12, K2CO3, MeCN; (c) TFA;
(d) Gd2O3, H2O, 50 ꢁC.
Upon incubation, all complexes enter into HTC cells in
amounts that appear definitively higher than the ones
that could be expected by simple pinocytosis (Fig. 1).
Table 1. Relaxivity of the gadolinium complexes studied
Compound
R1 (sꢀ1 mMꢀ1 a
)
Gd-DTPAGlu-Gln 7a
Gd-DTPAGlu-Agm 7b
Gd-DTPAGlu-Arg 7c
Gd-DOTA-Gln 10a
Gd-DOTA-Agm 10b
Gd-DOTA-Arg 10c
Gd-DTPAGlu 13
6.5
6.8
6.8
5.0
5.1
5.6
6.2
Internalisation (37˚C)
Blank
a Values obtained at 20 MHz and 25 ꢁC.
* *
*
37% HCl and sonicated at 120 ꢁC. After 16 h, all Gd3+ is
dissolved as free aqua-ion.
By measuring the proton relaxation rate (R1obs) of these
solutions it is possible to determine the Gd3+ concentra-
tion. In fact, R1obs is proportional to the concentration
of the paramagnetic Gd3+ ion according to the formula:
˚
˚
˚
R1obs ¼ R1d þ ½Gd3þꢁ ꢂ rGd3þ
where R1d is the relaxation rate obtained with the same
Membrane binding (4˚C)
1p
Gd3þ
number of untreated cells, and r
the millimolar
1p
Figure 2. MRI images of cellular pellets in agar (7 T). The HTC ( )
*
relaxivity of the Gd ion (13.5 sꢀ1 mMꢀ1 in a 6 M HCl
solution). The method has been checked out by using
atomic standard solutions of Gd3+ ion and double-
checked with parallel ICP-MS measurements.
and hepatocytes (ꢁ) have been incubated in EBSS medium added with
Gd-DTPAGlu-Gln 7a. T1 of HTC pellets: blank = 1.99 (s), bind-
ing = 1.15 (s), uptake = 0.96 (s). T1 of hepatocyte pellets: blank = 1.64
(s), binding = 1.65 (s), uptake = 1.45 (s).

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L. Lattuada et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4111–4114
In fact the latter process, that leads to a nonspecific
internalization of portions of the incubation medium,
may yield to significant entrapments only at concentra-
tion of imaging agents much higher than that those used
in the present work. Thus, on the basis of the amounts
of internalized Gd, one may conclude that the internal-
ization path involves the transport system of choice. By
comparing the MR images of HTC pellets incubated at
4 ꢁC with those incubated at 37 ꢁC (Fig. 2) one may
assess the relative effects on the attainable contrast
arising from the simple binding on the outer cellular
membrane and the overall uptake process. At low
temperature (4 ꢁC), the energy-dependent pathways
involved in the cellular uptake are not activated and
therefore the observed signal enhancement is limited to
the small amount of paramagnetic agent bound on the
outer cell membrane. Support to the view that glutamine
transporters are involved in the uptake process has been
gained by measuring a decrease in the amounts of inter-
nalized gadolinium complexes 7a when free glutamine
was added to the incubation medium (Fig. 3). Clearly
the free glutamine binds better to its transporter and
causes a significant decrease of the cellular uptake of
7a. On the basis of the available results it is not possible
to say anything concerning the eventual changes in the
internalization pathway of the amino acid-functional-
ized agents in respect to the free amino acids. Complex
7a gave the best performance being internalized to an
extent of about 5 · 109 Gd atoms per HTC cell. The
observation that complex 10a does not show an
analogous uptake outlines how the structure of the
chelate (DTPA vs DOTA), the residual charge, and
the nature of the spacer may be important in determin-
ing the efficiency of the uptake. Moreover, one or more
of these parameters, together to the increased pinocytic
activity of tumor cells, could be responsible for the
different HTC uptake of compounds 7b, 7c, and 13.
improved relaxometric properties in respect to the par-
ent non-conjugated complexes. Moreover, the complex
7a is able to recognize the trans-membrane glutamine
transport system and the in vitro experiments show that
the amount of internalized Gd is sufficient for the MRI
visualization of tumor cells in respect to healthy ones.
Further studies are ongoing with compound 7a on other
tumor cell lines.
Acknowledgment
This work was supported in part by the Ministero
`
dell’Istruzione, dell’Universita e della Ricerca of Italy.
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In summary, the results reported in this work show that
the conjugation of amino acids to DTPA and DOTA
Gd(III) complexes leads to products that display
4
3,5
3
Gd-DTPAGlu 13
Gd-DTPAGlu-Gln 7a
2,5
2
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1,5
1
20. The cells were grown in 75 cm2 flasks, in a humidified CO2
incubator, at 37 ꢁC, under air/CO2 95/5 atmosphere in
DMEM–F12 medium supplemented with 5% fetal bovine
serum (FBS), 100 U/ml penicillin, and 100 mg/ml
streptomycin.
0,5
0
0
2
4
6
8
10
21. Parameters used: repetition time (TR) = 10,000 ms; echo-
time (TE) = 3.3 ms; inversion-time (TI) = 1000 ms; num-
ber of excitation (NEX) = 1; FOV = 1 · 1 cm; data
matrix = 128 · 128; slice thickness = 1 mm.
Glutamine (mM)
Figure 3. Competition assay between complexes 7a and 13 and
glutamine for the uptake into HTC cells (6 h, 37 ꢁC, complex
concentration 1.3 mM).
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