K. Prinsen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3216–3220
3219
containing >95% of the 68Ga activity, was mixed with 37% HCl in order to
form [68GaCl4]ꢂ which was immobilized on an anion exchanger. Metallic
impurities and 68Ge were removed by elution of the anion exchange column
with 3.0 mL of 5.5 M HCl. Subsequently, 68Ga was eluted from the anion
exchanger using 0.8 mL of ultrapure water, yielding a low volume and highly
pure and concentrated solution of 68Ga which was used for radiolabeling. For
radiolabeling, the pH of the 68Ga solution was adjusted to pH 4–4.5 by addition
of a 0.5 M sodium acetate buffer (0.75 mL). After addition of 20 nmol bis-
whether the second chelator, accounting for the negative charge of
the tracer, is indeed essential for the selectivity of the tracer.
Acknowledgments
The authors would like to thank Peter Vermaelen and Ann Van
Santvoort for their skillful help with the animal experiments, Ellen
Devos for her help with the histochemical stainings and Ivan San-
nen for his help with the LC–MS analysis. Research funded by a
Ph.D. Grant of the Institute for the Promotion of Innovation
through Science and Technology in Flanders (IWT-Vlaanderen).
This research was further supported by the DiMI Project DiMI-
EU-FP6: SHB-CT-2005-512146 and the European Union (project
COST D38).
DOTA-BI (24 lL of a 1 mg/mL solution in ultrapure water), the labeling mixture
was heated at 90 °C for 10 min. For radio–LC–MS experiments, a carrier-added
radiolabeling was performed. For this purpose the amount of precursor was
increased to 0.2 lmol and an equal amount of non-radioactive gallium
(
69,71GaCl3) in 0.1 M HCl was added to the radiolabeling mixture. Purification
of the 68Ga-bis-DOTA-BI complex was carried out by passing the labeling
mixture over a Sep-PakÒ light C18 cartridge (Waters). After rinsing the Sep-Pak
column using 3.0 mL water, 68Ga-bis-DOTA-BI was eluted with 0.8 mL ethanol
and collected in a septum-sealed glass vial. After evaporation of the ethanol
with a stream of nitrogen, 68Ga-bis-DOTA-BI was resuspended in phosphate
buffered saline (PBS) and analyzed by RP-HPLC on an XTerraÒ RP C18 column
(5
lm, 4.6 mm ꢁ 250 mm; Waters) eluted with gradient mixtures of 0.1% TFA
References and notes
in H2O/0.1% TFA in acetonitrile (0 min: 100/0 v/v, 20 min: 10/90 v/v, linear
gradient) at a flow rate of 1.0 mL/min (tR = 11.8 min). Accurate mass radio–LC–
MS analysis (ESI-MS) for C57H71N14O16Ga [M+H]+: found: 1278.4530 Da, calcd
1278.4620 Da (tR = 10.4).
18. In vivo stability of 68Ga-bis-DOTA-BI was studied in normal NMRI mice by
determination of the relative amounts of intact parent tracer and radiolabeled
metabolites in plasma using RP-HPLC. The mice were anesthetized (isoflurane)
and intravenously injected with the tracer via a tail vein. At 10, 30, 90 or
240 min pi, the animals were sacrificed under anesthesia (isoflurane) by
decapitation (n = 2 per time point), blood was collected in a BD vacutainerÒ
tube (containing lithium heparin; Beckton–Dickinson, Franklin Lakes, USA) and
centrifuged at 3000 rpm (1837ꢁg) for 5 min (Eppendorf centrifuge 5810) in
order to separate plasma. Plasma (0.5 mL) was analyzed by RP-HPLC using a
ChromolithÒ Performance RP-18e column (3.0 mm ꢁ 100 mm; Merck,
Darmstadt, Germany) that was eluted with gradient mixtures of H2O
(solvent A), 0.1% TFA in H2O (solvent B) and 0.1% TFA in acetonitrile (solvent
C) (0–4 min: 100% A, 4–20 min: 100% B v/v to 10% B and 90% C v/v, linear
gradient) at a flow rate of 1.0 mL/min. The eluate was collected in 1.0-mL
fractions using an automatic fraction collector and the 68Ga activity in all
fractions was measured using an automatic gamma counter.
19. Evaluation of the uptake of 68Ga-bis-DOTA-BI in necrotic tissue was performed
by ex vivo autoradiography in rats with a reperfused partial liver infarction.
Animals were intravenously injected with 18.5–37 MBq 68Ga-bis-DOTA-BI via
a tail vein under anesthesia (isoflurane). The animals were sacrificed at 30 min
or 90 min pi (n = 3 per time point) by an intraperitoneal overdose of
pentobarbital. The liver was removed and thoroughly washed with 0.9%
saline (4 °C) to remove blood pool activity. The different liver lobes were
separated and stained in 1.5% (w/v) triphenyltetrazolium chloride solution in
0.9% saline for 10 min at 37 °C. Guided by the TTC staining, the necrotic and
viable liver tissue were identified and samples were then rapidly frozen in
isopentane cooled with liquid nitrogen to ꢂ50 °C. The frozen tissue was cut
immediately afterwards with a cryotome (Shandon Cryotome FSE; Thermo
16. Compound 2. To
a solution of ethyl 1H-indole-2-carboxylate (1) (1.89 g,
10 mmol) in ethanol (20 mL), benzaldehyde (0.53 g, 5 mmol) and 37% HCl
(0.4 mL) were added under nitrogen atmosphere. The reaction mixture was
heated to reflux and stirred for 2 h followed by cooling to room temperature
(rt). The precipitate from the cooled solution was filtered, washed with cold
ethanol and dried over MgSO4. Yield: 2.076 g (4.49 mmol; 89%). 1H NMR (400-
MHz, DMSO-d6) d 11.72 (s, 2H, NH indole), 7.44 (d, 2H, Ph), 7.37 (s, 1H, Ph-CH),
7.24 (m, 3H, Ph), 7.06–7.14 (m, 4 H, Ph), 6.45–6.66 (m, 4H, Ph), 4.19 (q, 4 H,
CH2), 1.17 (t, 6H, CH3). ESI-MS for C29H26N2O4 [M+Na]+: found 489.18 Da,
Fisher, Waltham, USA) into 10-, 30- and 50-lm serial sections which were
thaw-mounted on glass slides. The sections were exposed to a super resolution
screen (Canberra-Packard, Meridan, USA) overnight and digital
autoradiographic images were obtained by reading out the phosphor screen
using a Cyclone Phosphor Imager scanner (Canberra-Packard). Images were
analyzed using Optiquant software (version 5.0; Canberra-Packard). After
exposure for autoradiography, the sections were stained with hematoxylin and
eosin (H&E) using a standard procedure and examined under a microscope.
calculated 489.17 Da. Compound 3: compound
2 (0.5 g, 1.07 mmol) and
hydrazine monohydrate (1 mL, 20.6 mmol) were dissolved in a mixture of
pyridine (6 mL) and methanol (3 mL) and the resulting mixture was heated to
reflux and stirred for 48 h. After removal of solvents and excess hydrazine
azeotropically with water under reduced pressure, the residue was dissolved in
dichloromethane and the hydrazide was precipitated by addition of
acetonitrile to the solution. The white precipitate was collected by filtration
and dried over MgSO4. Yield: 0.34 g (0.78 mmol; 73%). 1H NMR (400-MHz,
DMSO-d6) d 11.38 (s, 2H, NH indole), 9.57 (s, 2H, CONH), 7.39 (d, 2H, Ph), 7.25
(s, 1H, Ph-CH), 7.21 (m, 3H, Ph), 6.99–7.09 (m, 4H, Ph), 6.58–6.66 (m, 4H, Ph),
4.5 (s, 4H, NH2). ESI-MS for C25H22N6O2 [M+Na]+: found 461.17 Da, calculated
20. MicroPET imaging was performed on
a FOCUS 220 tomograph (Siemens/
Concorde Microsystems, Knoxville, USA). Rats with a reperfused partial liver
infarction (n = 3) were anesthetized with 1.5–2.5% isoflurane in O2 at a flow
rate of 1–2 L/min and fixed in prone position on the bed of the microPET
tomograph. Dynamic microPET images were acquired for 240 min with a mean
activity of 74.4 11.8 MBq 68Ga-bis-DOTA-BI after intravenous administration
of the tracer via a tail vein as a bolus injection. The microPET listmode data
were histogrammed into 51 frames (4 ꢁ 15 s, 4 ꢁ 60 s, 5 ꢁ 180 s, 8 ꢁ 300 s, and
461.17 Da. Compound 4: compound
3 (17.5 mg, 0.04 mmol), DOTA-NHS
30 ꢁ 360 s) and reconstructed using
a 2D OSEM algorithm before being
(50 mg, 0.1 mmol) and DIEA (77.5 L, 0.45 mmol) were dissolved in dry DMF
l
analyzed using PMOD (version 2.65; PMOD, Zurich, Switzerland). Volumes of
interest were created on mean images in order to perform a quantitative
analysis and generate time–activity curves (TACs) of the tracer in normal and
necrotic liver tissue. After acquisition of the microPET images, the rats were
sacrificed by an intraperitoneal overdose of pentobartibal to perform ex vivo
autoradiography in correlation with histochemical analysis as described above.
21. Distribution of 68Ga-bis-DOTA-BI in mice with Fas-induced hepatic apoptosis
and control mice was measured in order to investigate the selectivity of the
tracer for necrotic cell death. The biodistribution studies in FasL treated (n P4;
90 min pi of FasL) and untreated control mice (n P4) were performed 60 min
pi of the tracer. Mice were anesthetized with isoflurane and 370 kBq of the
tracer was injected via a tail vein. The mice were sacrificed by decapitation
under anesthesia (isoflurane) 60 min pi of the tracer. A biodistribution study
was performed as described above and the hepatic uptake of the tracer was
expressed as % ID/g. Immediately after quantification of 68Ga activity, the livers
of FasL treated and untreated control mice were fixed in 6% formaldehyde.
(2 mL) and the resulting mixture was stirred for 24 h at rt. After addition of
water the solvents were removed under reduced pressure and the resulting
white powder was dissolved in a mixture of acetonitrile/H2O (1:1, v/v) and
purified by preparative RP-HPLC using an XTerraÒ Prep RP18 column (10
lm,
10 mm ꢁ 250 mm; Waters) eluted with gradient mixtures of 0.1% TFA in H2O/
0.1% TFA in acetonitrile (0–25 min: 80/20 to 35/65 v/v, linear gradient) at a
flow rate of 3.0 mL/min. Yield: 34 mg (0.028 mmol; 70%). 1H NMR (600-MHz,
DMSO-d6) d 11.79 (br s, 6H, COOH), 10.21 (br s, 2H, NH indole), 7.42 (m, 2H,
Ph), 7.21 (m, 3H, Ph), 7.16 (m, 1H, Ph-CH), 7.14 (m, 2H, Ph), 6.98 (m, 2H, Ph),
6.65 (m, 2H, Ph), 6.58 (m, 2H, Ph), 3.84–3.70 (m, 4H, COCH2N), 3.48–3.29 (m,
12H, NCH2COOH), 3.03 (m, 32H, CH2N). 13C NMR (125-MHz, DMSO-d6) d
162.19, 160.15–159.50, 144.23, 136.13, 128.97, 128.37, 127.18, 126.75, 124.87,
124.53, 122.75, 121.97, 120.18, 112.93, 55.68, 50.49 and 46.33. ESI-MS for
C
57H74N14O16 [M+H]+: found 1211.45 Da, calculated 1211.54 Da.
17. Elution of the 68Ge/68Ga generator, purification and concentration of the eluate
was carried out as previously reported [24,25]. Briefly, 68Ga was eluted from
the generator in three fractions using 0.1 M HCl. The second fraction,
After paraffin embedding, 5-lm thick sections were cut, stained with H&E and
Prinsen, Kristof
