Synthesis and evaluation of a new 99mTc(I)-tricarbonyl complex bearing the 5-nitroimidazol-1-yl moiety as potential hypoxia imaging agent

Article abstract of DOI:10.1002/jlcr.3195

The objective of this work was to develop a novel ^99mTc complex bearing the 5-nitroimidazol-1-yl moiety with recognised selectivity towards hypoxic tissue, as a potential radiopharmaceutical for imaging tumour hypoxia.The new metronidazole derivative (2-amine-3-[2-(2-methyl-5-nitro-1H- imidazol-1-yl)ethylthio]propanoic acid) (L) containing adequate groups to coordinate technetium through the formation of a Tc(I)-tricarbonyl complex was synthesised with adequate yield (33%) and characterised by spectroscopy. Labelling was performed by substitution of three labile water molecules of the technetium tricarbonyl precursor, fac-[^99mTc(CO)₃(H ₂O)₃]⁺ with the ligand. A radiochemical purity higher than 90% was achieved and remained unchanged for more than 4 h. The complex has a high stability in plasma, a moderate plasma protein binding and a moderate hydrophilicity.In vitro cell uptake studies showed a ratio between the activity taken up by cells in hypoxia/normoxia of 1.6 ± 0.4 (p < 0.5).Biodistribution in normal mice showed rapid depuration and low uptake in all organs and tissues except liver. Biodistribution in mice bearing induced tumours showed a low tumour uptake, but tumour/muscle ratio was favourable thanks to depuration. Comparison with biological results of other metronidazole derivatives clearly shows that modifications of the chelator are very important and contribute to improve the biological behaviour.

Full text of DOI:10.1002/jlcr.3195

Research Article
Received 9 May 2013,
Revised 9 December 2013,
Accepted 9 February 2014
Published online 1 April 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3195
99m
Synthesis and evaluation of a new Tc(I)-
tricarbonyl complex bearing the 5-
nitroimidazol-1-yl moiety as potential
hypoxia imaging agent
a
b
a
c
a
*
J. Giglio, S. Dematteis, S. Fernández, H. Cerecetto, and A. Rey
The objective of this work was to develop a novel 99mTc complex bearing the 5-nitroimidazol-1-yl moiety with recognised
selectivity towards hypoxic tissue, as a potential radiopharmaceutical for imaging tumour hypoxia.
The new metronidazole derivative (2-amine-3-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]propanoic acid) (L) containing
adequate groups to coordinate technetium through the formation of a Tc(I)-tricarbonyl complex was synthesised with
adequate yield (33%) and characterised by spectroscopy.
Labelling was performed by substitution of three labile water molecules of the technetium tricarbonyl precursor, fac-
[
99mTc(CO)3(H2O)3]+ with the ligand. A radiochemical purity higher than 90% was achieved and remained unchanged
for more than 4 h. The complex has a high stability in plasma, a moderate plasma protein binding and a moderate
hydrophilicity.
In vitro cell uptake studies showed a ratio between the activity taken up by cells in hypoxia/normoxia of 1.6 0.4 (p< 0.5).
Biodistribution in normal mice showed rapid depuration and low uptake in all organs and tissues except liver.
Biodistribution in mice bearing induced tumours showed a low tumour uptake, but tumour/muscle ratio was favourable thanks
to depuration. Comparison with biological results of other metronidazole derivatives clearly shows that modifications of the
chelator are very important and contribute to improve the biological behaviour.
Keywords: Tc-tricarbonyl complexes; 5-nitroimidazole derivatives; hypoxia imaging agent
amine and thiol group), which acts as tridentate ligand towards the
Introduction
Tc(I)- tricarbonyl core.
In order to assess the potentiality of the 99mTc-complex as
Tumour perfusion and oxygenation status have been recognised
as important factors that may cause poor treatment outcome
hypoxia-targeting radiopharmaceutical, we have studied its main
physicochemical and biological properties both in vitro and
in vivo.
after radio and chemotherapy.1,2
Bioreductive compounds, which are selectively reduced in hypoxic
tissue to reactive intermediates that bind to intracellular molecules,
have been used for the development of potential radiopharma-
Materials and methods
ceuticals for targeting hypoxic tumours. The nitroimidazol-1-yl
moiety is one of the preferred pharmacophores,3–8 but other
General
functional groups as nitroaromatics9,10 and N-oxides11 have also
been studied. However, properties of the previously studied
technetium complexes are not yet ideal, and new potentially active
compounds are still being developed.
All laboratory chemicals were reagent grade and were used without
further purification. Solvents for chromatographic analyses were
The 5-nitroimidazole metronidazole (Figure 1), which has
demonstrated high affinity for hypoxic tumours in vitro and in vivo
aCátedra de Radioquímica, Facultad de Química, Universidad de la República,
is an adequate starting material for the preparation of 99mTc Montevideo, Uruguay
radiopharmaceuticals.12–15 Metronidazole should be attached to
bCátedra de Inmunología, Facultad de Química, Universidad de la República,
Montevideo, Uruguay
adequate chelating groups to enable coordination of 99mTc without
losing the biological activity. As part of our ongoing research on
potential radiopharmaceuticals for imaging tumour hypoxia,9–11,16–19
we have designed and synthesised the novel metronidazole
derivative 2-amine-3-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]
propanoic acid (L) (Figure 1). Chemical modifications were performed
cÁrea de Radiofarmacia y Radioquímica-CIN, Facultad de Ciencias, Universidad
de la República, Montevideo, Uruguay
*Correspondence to: Dra. Ana Rey, Facultad de Química, General Flores 2124,
11800 Montevideo, Uruguay.
in order to introduce a cysteine-like donor-atom set (carboxylate, E-mail: arey@fq.edu.uy
J. Label Compd. Radiopharm 2014, 57 403–409
Copyright © 2014 John Wiley & Sons, Ltd.

J. Giglio et al.
Synthesis of 2-amine-3-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]
propanoic acid (L)
Synthesis was performed according to a previously described
method.21 A solution of the intermediate compound 1 (0.2 g,
0.71 mmoles) dissolved in THF (10 mL) was added to a solution
of cysteine hydrochloride (0.125 g, 0.71 mmoles) dissolved in
triethylamine (0.10 mL). Progress of the reaction was monitored
by TLC until the total consumption of the intermediate
compound (approximately 24 h). A white solid corresponding
to the impurity triethylamine hydrochloride was removed by
filtration. The solvent was evaporated under vacuum to yield
Figure 1. Metronidazole and final ligand L structures.
HPLC grade. [99mTc]NaTcO4 was obtained from a commercial
generator (Tecnonuclear S.A., Argentina). Carbonyl labelling
agent Isolink was provided by Covidien (Dublin, Ireland). NMR
spectra were obtained in the indicated deuterated solvent using
a Bruker Avance DPX 400 Spectrometer (Billerica, MA, USA).
Chemical shifts are reported as δ values (parts per million)
relative to residual protons of deuterated solvent. Coupling
constants are reported in hertz (Hz). The multiplicity is defined
1
the desired product. White solid (70%); mp = 90–94 °C. H NMR
(CDCl3) δ (ppm): 1.33(m, 2H), 2.57 (s, 3H), 3.27 (m, 1H), 3.57
(t, 2H, J = 14.2 Hz), 4.46 (t, 2H, J = 14.2 Hz), 7.98 (s, 1H). 13C NMR
(CDCl3) δ (ppm): 14.9 (CH3), 36.5 (CH2), 46.0 (CH2), 51.4 (CH2),
62.4 (CH), 129.0 (CNO2), 133.9 (CH), 151.7 (C), 182 (COOH). IR (KBr)
ν (cmÀ1): 1386, 1528, 1732, 3622. Mass spectra (electron impact,
70 eV), m/z: 275 (M+ + H); 244 (M+ À NO); 199 (MÀ NOÀ CO2H).
by
s (singlet), t (triplet) or m (multiplet). Thin layer
chromatography (TLC) was carried out on precoated plates of
silica gel 60 F254. Flash chromatography was performed using
silica gel 60 (230–400 mesh, Sigma-Aldrich, St. Louis, MO,
USA). For column chromatography, we used silica gel (60–230
mesh, Merck (Merck & Co., Inc., NJ, USA)). Melting points were
Radiolabelling
determined with an electrothermal melting point apparatus Preparation of fac-[99mTc(CO)3(H2O)3]+ complex using a kit formulation
(Electrothermal 9100 Staffordhire, UK.) and were uncorrected.
The 99mTc-sodium pertechnetate (185–1850 MBq, 1 mL) was
The mass spectra were conducted with a mass spectrometer,
added to an Isolink kit (Covidien, Dublín, Ireland), and the
Hewlett Packard 5973 MSD or MICROMASS (Triple Quattro Palo
mixture was incubated at 100 °C for 30 min. After cooling, pH
Alto, CA, USA.), using electron impact or electrospray (ESI),
was adjusted to 7.0 with 1N HCl solution. Complex formation
respectively. HPLC analysis was carried out on an LC-10 AS
was monitored by HPLC analysis as indicated in the general
Shimadzu Liquid Chromatography System using a reverse
experimental section. Retention time was 4.0 min. Radiochemical
phase column Phenomenex Luna 5 μm, C18 column
purity was >90%.
(4.6 × 150 mm). Elution was performed with a binary gradient
system at 1.0 mL/min flow rate using triethylamine-phosphate
Preparation of fac-[99mTc(CO)3(H2O)3]+ complex using CO(g)
buffer pH 2.5 as mobile phase A and methanol as mobile
The 99mTc-precursor complex was prepared according to a
phase B; the elution profile was as follows: 0–3 min 100 %
previously described method22 as follows: Na/K tartrate
A; 3–6 min linear gradient to 25% B; 6–9 min linear gradient
(20.0 mg), Na2CO3 (4.0 mg) and NaBH4 (7.0 mg) were placed in
to 34% B; 9–20 min linear gradient to 100% B; 20–27 min
a vial. The vial was sealed and flushed with carbon monoxide
100% B; 27–30 min linear gradient 0% B. Detection
for 30 min. 99mTc-sodium pertechnetate (185–1850 MBq, 1 mL)
was accomplished either with a photodiode array detector
was added, and the mixture was incubated at 75 °C for 30 min.
(SPD-M10A, Shimadzu (Shimadzu Corporation, Kyoto, Japan))
After cooling, pH was adjusted to 7.0 with 1N HCl solution. The
that recorded UV-vis spectra on flux or with a ‘3 × 3’ NaI (Tl)
complex formation was monitored by HPLC analysis as indicated
crystal scintillation detector. Activity measurements were
in general experimental section. Retention time was 4.0 min.
performed either in a Dose Calibrator, Capintec CRC- 5R
Radiochemical purity was >90%.
(Ramsey, NJ, USA) or in a scintillation counter, ‘3 × 3’ NaI (Tl)
crystal detector associated to an ORTEC (Atlanta, USA)
monochannel analyser.
Substitution with L
Neutralised fac-[99mTc(OH2)3(CO)3]+ precursor (100–200 μL,
185–1850MBq) was incubated with ligand L (4mg, 1 × 10À5 mol)
for 30min at 70 °C. Complex formation was monitored by HPLC
analysis. Retention time was 14.0min. Radiochemical purity
was >90%.
Synthesis
Synthesis of 1-(2-iodoethyl)-2-methyl-5-nitro-1H-imidazole (1)
Synthesis was performed according to a previously described
method.20 Triphenylphosphine (0.896 g, 3.42 mmoles) and imida-
zole (0.233 g, 3.42 mmoles) were dissolved in dry dichloromethane
(10 mL). Iodine (0.869 g, 3.42 mmoles) was slowly added to the
solution. After incubation at room temperature during 5 min, a
solution of metronidazole (0.4g, 2.34 mmoles) in dichloromethane
(1.0mL) was added to the mixture. Reaction was controlled by TLC
until total consumption of metronidazole (approximately 24h).
The solvent was evaporated under vacuum. The residue was
Physicochemical evaluation
Stability in labelling milieu
99mTc-complex was incubated in the labelling milieu at room
temperature, and the radiochemical purity was assessed by
HPLC using the chromatographic conditions described in
general experimental section for up to 4 h after labelling.
Stability in plasma
purified by column chromatography (SiO2, dichloromethane/
1
methanol 85:15). Brown solid (47%); mp= 78 °C. H NMR (CDCl3) 99mTc-complex (100 μL) was incubated in human plasma (900 μL)
δ (ppm): 2.60 (s, 3H), 3.48 (t, 2H, J = 12.0 Hz); 4.65 (t, 2H, at 37 °C for up to 4 h. After 30, 60 and 120-min incubation,
J = 12.0Hz), 8.00 (s, 1H). IR (KBr) ν (cmÀ1): 1355, 1368, 1545, 505.
samples (200 μL) were extracted and proteins were precipitated
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Copyright © 2014 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2014, 57 403–409

J. Giglio et al.
with ethanol (200 μL), centrifuged (12000 rpm, 5 min) and the were not weighed. Corrections by different sample geometry
supernatants analysed by HPLC using chromatographic conditions were applied when necessary. Results were expressed as %
described in general experimental section.
dose/organ. % Dose in blood and in muscle were calculated
considering the whole blood as 7% and the whole muscle
as 43% of the body weight, respectively
Protein binding studies
99mTc-complex (25 μL) was incubated with human plasma
(475 μL) at 37 °C for up to 120 min. At 30 and 120-min aliquots
(50 μL) were added to MicroSpin G-50 columns (GE Healthcare
Buckinghamshire, UK.), which have been pre-spun at 2000 g for
1 min. Columns were centrifuged again at 2000 g for 2 min,
and the collected eluate and the column were counted in a
NaI(Tl)-scintillation counter. Protein bound tracer was calculated
as the percentage of activity eluted from the column.
Biodistribution in animals bearing induced tumours
A culture of 3LL Lewis murine lung carcinoma cells was
expanded and treated with trypsin previous to inoculation. A cell
suspension in PBS containing 3 × 106 cells was prepared and
injected subcutaneously in the right limb of C57BL/six mice
(8–10 weeks old). About 20–30 days later, the animals
developed palpable tumour nodules (1.5 × 0.5 × 0.5 cm) and
were used for biodistribution studies.
Three animals per group were injected via a lateral tail with
99mTc-L (0.1 mL, 0.037–0.37 MBq). At different intervals after
injection, the animals were sacrificed by neck dislocation. Whole
tumour and samples of blood and muscle were collected,
weighed and assayed for radioactivity. Results were expressed
as % dose/organ and % dose/g tissue.
Lipophilicity
Lipophilicity was studied through the apparent partition
coefficient between 1-octanol and phosphate buffer (0.125 M,
pH 7.4). In a centrifuge tube, containing 2 mL of each phase,
0.1 mL of the 99mTc complex solution was added, and the
mixture was shacked on a Vortex mixer and finally centrifuged
at 5000 rpm for 5 min. Three samples (0.2 mL each) from each
layer were counted in a gamma counter. The partition coefficient
was calculated as the mean value of each cpm/mL of 1-octanol
layer divided by that of the buffer. Lipophilicity was expressed
as log P.
Results and discussion
Synthesis of the ligand
Design of the ligand consisted in the introduction to
metronidazole of a high-affinity tridentate chelator system to form
a stable neutral complex with the [99mTc(CO)3(H2O)3]+ synthon. For
this purpose, we have prepared a cysteine-derivative ligand that
bears an NSO type-chelator unit, similar to the homocysteine-
derivative developed by Karagiorgou et al.,23 in which the donor-
atom system consists on an N-primary amine, an S-thioether and
an O-carboxylate.
Synthesis of the ligand was achieved by a two-step reaction
using metronidazole as starting reagent (Figure 2). In the first step,
the intermediate compound 1 was obtained by substitution of the
hydroxyl group of metronidazole by an iodine moiety. In the
second step, attachment to a cysteine unit was performed in an
organic solvent and alkaline medium. Intermediate and final
products were purified and characterised by common spectro-
scopic techniques (NMR, IR) and mass spectrometry.
Biological evaluation
Cell uptake studies
The cell culture studies were performed using the adherent cell
line HCT-15 (CCL-255TM ATCC) corresponding to human adeno-
carcinoma. Cells were cultured in RPMI-1640 (R6504 Sigma-
Aldrich) supplemented with 10% foetal bovine serum (Gibco),
penicillin 100 U/mL (Sigma) and 100 μg/mL streptomycin (Sigma)
in T75 tissue culture flasks (Nunc, Denmark) at 37°C and 5% CO2
until approximately 7.5× 106 cells were obtained. Afterwards, the
flasks were pre-incubated in a chamber gassed with N2 for 1 h to
remove oxygen from the milieu, and afterwards 99mTc-complex
was added and incubated for additional predefined incubation
times (60–120 min). The same procedure was repeated in normal
culture conditions (37 °C and 5% CO2) to use as control. After
incubation, time-elapsed culture milieu was removed, cells were
washed with PBS and treated with Trypsin-EDTA (Sigma). Finally,
Radiolabelling
activity in the supernatant and the cells were measured in a solid Synthesis of the 99mTc-tricarbonyl complex was achieved by a
scintillation counter, and results were expressed as the ratio two-step synthetic route (Figure 3). The first step consisted in
between the percentage of activity taken up by cells incubated the preparation of the precursor, fac-[99mTc(CO)3(OH2)3]+, either
in nitrogen (hypoxia) and incubated in normal conditions using sodium pertechnetate eluted from a 99Mo/99mTc generator
(normoxia).
as starting material and sodium borohydride as reducing agent
Animal studies
All animal studies were approved by the Ethics Committee of the
Faculty of Chemistry from Uruguay.
Biodistribution in normal mice
CD1 mice (female mice, 25–30 g, three animals per group)
were injected via a lateral tail vein with 99mTc-L (0.1 mL,
0.037–0.37 MBq). At different intervals after injection, animals
were sacrificed by neck dislocation. Whole organs and
samples of blood and muscle were collected, weighed and
assayed for radioactivity. The total urine volume was
collected during the biodistribution period and also removed
from bladder after sacrifice. The bladder, urine and intestines
Figure 2. Synthesis of the ligand L.
J. Label Compd. Radiopharm 2014, 57 403–409
Copyright © 2014 John Wiley & Sons, Ltd.
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J. Giglio et al.
Figure 3. Radiolabelling route.
Figure 4. Typical chromatograms for fac-[99mTc(CO)3(OH2)]+ (a) and [99mTc(CO)3(L)] (b) (RP-HPLC).
Physicochemical evaluation
in the presence of CO(g) or, as an alternative, the Isolink kit. The
second step involved the substitution of the precursor by
incubation with the ligand, L. It is well known that S-alkylated
cysteine derivatives bind efficiently with the tricarbonyl core
via its NSO donor-atom set to provide stable and neutral
complexes. Coordination affords two five-membered and one
six-membered ring providing thermodynamically stable
hexacoordinated complexes23–26 . The proposed structure for
the final complex is shown in Figure 3.
In order to study the potentiality of the 99mTc-complex as
radiopharmaceutical, some relevant physicochemical properties
were studied (i.e. stability, lipophilicity and protein binding).
Stability
The final complex was stable for at least 4 h in labelling milieu at
room temperature. Besides, stability after incubation with human
plasma for 4 h at 37°C was also studied, and radiochemical purity
after incubation was found to be 91.0 2.0%.
Radiochemical purities of precursor fac-[99mTc(CO)3(OH2)3]+
and the final complex [(99mTc(CO)3(L)] were evaluated by reverse
phase HPLC (RP-HPLC, Figure 4). The precursor was obtained
with high radiochemical purity (>90%). Substitution yielded a
single radiolabelled species with retention time of 14 min
(Figure 4b) and radiochemical purity above 90%.
Protein binding studies
Binding to plasma protein was studied using size exclusion
chromatography. Ideally, low protein binding is required in order
Table 2. Biodistribution results of [99mTc(CO)3(L)] in C57BL/
six mice bearing induced Lewis murine lung carcinoma. Each
value represents the mean percentage (n = 3) of the injected
dose/organ standard deviation of the mean
Table 1. Biodistribution results of [99mTc(CO)3(L)] in normal
mice. Each value represents the mean percentage (n = 3) of
the injected dose/organ standard deviation of the mean
% Injected dose/organ
% Injected dose/organ
Organ
0.5 h
2.0 h
4.0 h
Organ
0.5 h
2.0 h
4.0 h
Blood
Liver
Heart
Lung
8.20 0.98
35.5 4.1
0.40 0.09
0.61 0.09
0.54 0.31
11.8 3.0
0.13 0.05
5.4 2.7
1.40 0.09
1.68 0.58
23.0 6.0
26.7 5.4
2.07 0.03
19.7 2.3
2.00 0.18
21.9 4.3
Blood
Liver
Heart
Lung
7.2 1.5
38.1 6.5
0.32 0.12
0.48 0.05
0.71 0.12
12.9 3.9
0.09 0.03
4.1 3.2
1.98 0.17
3.45 0.89
25.8 8.0
22.5 8.2
2.53 0.12
22.3 1.1
0.45 0.17
0.09 0.02
0.14 0.08
6.8 2.1
0.02 0.01
3.26 0.92
0.83 0.13
0.92 0.32
28.5 5.1
34.3 4.3
1.85 0.32
20.2 3.4
0.12 0.09
0.08 0.05
0.12 0.06
5.92 0.23
0.02 0.01
1.92 0.27
0.43 0.21
0.14 0.12
30.2 9.2
38.0 8.1
0.09 0.03 0.09 0.08
0.16 0.03 0.13 0.09
0.08 0.04 0.15 0.03
5.42 1.06 5.16 0.44
0.02 0.01 0.02 0.01
2.26 0.80 1.80 0.28
0.97 0.62 0.66 0.13
0.61 0.12 0.39 0.21
Spleen
Spleen
Kidneys
Thyroid
Muscle
Gallbladder
Stomach
Intestines
Urine + bladder
Kidneys
Thyroid
Muscle
Gallbladder
Stomach
Intestines
Urine + bladder
27.6 2.7
39.9 6.8
32.1 7.8
35.0 5.3
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J. Giglio et al.
of the 99mTc complex. A log P of À0.75 0.08 was found. This
value is in accordance with the structure of the ligand,
because incorporation of the cysteine unit increases the
hydrophilicity of the ligand.
Table 3. Uptake in tumour of [99mTc(CO)3(L)] in C57BL/6
mice bearing induced Lewis murine lung carcinoma. Each
value represents the mean percentage (n = 3) of the injected
dose/g of tissue standard deviation of the mean
% Injected dose/g
Organ
Biological evaluation
or ratio
0.5 h
1.0 h
2.0 h
4.0 h
Cell uptake studies
In vitro uptake of [99mTc(CO)3(L)] both in normoxia and hypoxia
was evaluated using human colon adenocarcinoma HCT-15 cells
in culture. Cells were incubated at 37 °C under an atmosphere of
95% air plus 5% carbon dioxide (aerobic exposure) or 95%
nitrogen plus 5% carbon dioxide (hypoxic conditions). According
to literature, oxygen concentration ˂1000 ppm is considered
hypoxic condition.28,3 After 60 min equilibration period, the
radiolabelled compound was added and incubated with the cells
for other 60 min. HCT-15 cells maintain >90% of viability for at
least 90 min under the hypoxic conditions of this assay, as
demonstrated by the propidium iodide viability test.29 Finally,
cells were separated from supernatant and activity measured in
order to compare the percentage taken up in normoxic and
hypoxic conditions. This study has been previously validated by
our group and used successfully in the evaluation of other
Tumour
Blood
1.3 0.4 0.5 0.1 0.31 0.03
0.4 0.1
1.4 0.2
0.20 0.03
2.0 0.1
5.7 0.9 2.2 0.1
1.0 0.1 0.3 0.1
1.3 0.5 1.7 0.2
1.4 0.1
0.2 0.1
1.2 0.1
Muscle
Tumour/
muscle
Tumour/
blood
0.2 0.1 0.2 0.1
0.2 0.1
0.3 0.2
to ensure adequate pharmacokinetics of the potential radio-
pharmaceuticals. Additionally, only the unbound fraction of the
radiotracer will penetrate cells and other biological membranes.27
A relatively low protein binding of 15.0 5.0% was obtained for
[
99mTc(CO)3(L)], correlating with the high in vitro stability and
low lipophilicity of this complex. Because protein binding
remained constant during all the studied period, the reported
value corresponds to the mean of all observed values between
30 and 120 min.
[
99mTc]-complexes.17–19 Results for [99mTc(CO)3(L)] showed that
the ratio between hypoxic and normoxic uptake was 1.6 0.4.
This result is statistically significant (p ˂ 0.05) and confirms
selectivity towards hypoxia conditions.
Lipophilicity
Biodistribution in normal mice
The partition coefficient between 1-octanol and phosphate The in vivo behaviour was evaluated by biodistribution studies in
buffer (pH 7.4) was measured in order to assess lipophilicity normal mice at 0.5, 2 and 4 h post-injection. Table 1 shows the
Table 4. Some physicochemical and biological properties of our developed 99mTc(I)-tricarbonyl complexes
Physicochemical properties
Activity ina
Stability in
Protein
binding
Complex
plasma (%)a
log P
Blood
Liver
Kidneys
4.9 0.9
85
À0.82 0.02 51.5 0.9%
4.7 1.3 17.0 0.6
92
91
À0.44 0.04 13.0 3.0%
0.4 0.2
1.8 0.6 0.22 0.04
À0.75 0.08 15.0 5.0% 2.07 0.03 19.7 2.3 5.42 1.06
aPercentage of injected dose in each organ after 2 h of complex injection.
J. Label Compd. Radiopharm 2014, 57 403–409
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J. Giglio et al.
results expressed as percentage dose per organ in the most bearing an cysteine-like chelating unit, suitable to coordinate
significant organs as a function of time. [99mTc(CO)3(L)] showed 99mTc through a Tc(I)-tricarbonyl complex. Labelling with high
considerable blood uptake at 30 min post-injection but clearance radiochemical purity was successfully achieved, and the complex
was high after 1 h. Lung and muscle activities are low, and was stable both in labelling milieu and in human plasma.
moderate liver uptake, which decreases during time was also Lipophilicity and protein binding value were low. Biodistribution
observed. Excretion occurred through both the urinary and in normal mice was favourable, with rapid depuration and low
hepatobiliary tracts. Thyroid and stomach activities were very uptake in all organs and tissues except liver. Although cell
low, indicating minimal in vivo reoxidation. Uptake in other studies suggest
a preferential uptake by hypoxic tissue,
organs was negligible.
biodistribution in mice bearing hypoxic tumours showed low
uptake probably because of hydrophilicity. However, tumour/
muscle ratio was favourable thanks to depuration from soft
tissues. Comparison with other 99mTc tricarbonyl metronidazole
derivatives developed by our group leads to the conclusion that
not only ligand denticity but also lipophilicity plays a key role in
the biological behaviour.
Although selective cell uptake in hypoxic conditions was
observed and overall biodistribution in normal mice was
favourable, low uptake and scarce retention in tumour are
drawbacks of this compound as potential hypoxia imaging agent
in tumours.
Biodistribution in animals bearing induced tumours
In order to assess the potentiality of our approach for the design of
potential radiopharmaceuticals for nuclear oncology, evaluation of
[
99mTc(CO)3(L)] in C57BL/6 mice bearing tumours induced by
inoculation of 3LL Lewis murine lung carcinoma cells was
performed. Cells were inoculated subcutaneously in the right limb,
and biodistribution studies were performed 20–30 days after
inoculation, when tumours have adequate size. This animal
model was selected because histopathologic studies performed
previously by our group demonstrated high degree of hypoxia
within the tumours.17 Biodistribution results (Table 2) are in
full agreement to those obtained in normal CD1 mice.
Table 3 summarises the in vivo tumour uptake (expressed as
percentage dose per gramme) as well as the tumour/blood and
tumour/muscle ratios. [99mTc(CO)3(L)] showed relatively good initial
tumour uptake (1.3 0.4% dose gÀ1 at 0.5 h post-injection) although
approximately 50% of the activity was cleared from tumour after 1 h.
Uptake remained constant from 1 to 4 h post-injection. Muscle
clearance is fast, leading to a favourable tumour/muscle ratio at
Acknowledgements
The authors want to thank ANII and PEDECIBA for scholarships
for JG and SF, Gramón Bagó del Uruguay S.A. for providing
metronidazole, Covidien for providing Isolink kits, Dr. Ioannis
Pirmettis, NCSR Demokritos, Greece, Dra. María Moreno and Dr.
Alejandro Chabalgoity, Lab. Vacunas Recombinantes, Facultad
de Medicina, Universidad de la República, Uruguay.
4 h post-injection (2.0 0.1% dose
g
À1). Statistical analysis
demonstrated that uptake in tumour was significantly higher in
comparison with muscle (p= 0.05) at this time point.
Conflict of Interest
Comparison with other 99mTc-labelled metronidazole
derivatives previously described by our group16–18 is interesting.
In spite of having exactly the same pharmacophore, the
physicochemical and biological behaviour showed significant
differences according to the selected labelling methods. Even if
the labelling method is the same, difference in the donor atoms
set used for coordination seems to play a significant role.
Understanding these differences is a key point for tailoring the
biological behaviour of novel compounds.
The authors did not report any conflict of interest.
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