C.-C. Chang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4133–4139
Table 2
4137
from each phase and assayed for radioactivity with a gamma coun-
Biodistribution of Al18F–NOTA–BZA in C57BL/6 mice bearing B16F0 tumor after iv
injection of 3.7 MBq Al18F–NOTA–BZA. Values were presented as% ID/g (mean ± SD,
n = 3 at each time point)
18
ter. A logP of À1.96 ± 0.14 revealed that Al F–NOTA–BZA is more
hydrophilic compared with those compounds reported in the liter-
1
23
10 18
F-FBZA (1.70),4 and our previously
ature, e.g.,
I-IBZA (1.44),
1
23
16
123
Organ
0.25 h
0.5 h
1.21 ± 0.26
1 h
2 h
reported compound
IHPA/IHPP (À0.38/0.07).
I-Iochlonicotinamide (0.67)
and
I-
19
Blood
Heart
Lung
Liver
Stomach
S.Int
3.92 ± 0.88
1.25 ± 0.11
1.69 ± 0.34
1.97 ± 0.32
1.85 ± 0.29
1.53 ± 0.34
1.42 ± 0.21
1.34 ± 0.23
1.29 ± 0.29
0.86 ± 0.12
0.94 ± 0.18
6.67 ± 0.91
0.20 ± 0.02
7.31 ± 1.90
3.07 ± 0.56
0.72 ± 0.13 0.26 ± 0.09
0.39 ± 0.06 0.24 ± 0.06
0.50 ± 0.11 0.22 ± 0.05
0.79 ± 0.12 0.29 ± 0.08
0.67 ± 0.12 0.26 ± 0.07
0.65 ± 0.14 0.32 ± 0.06
0.30 ± 0.04
0.52 ± 0.14 0.23 ± 0.07
0.51 ± 0.11 0.17 ± 0.04
0.10 ± 0.03
0.68 ± 0.18 0.39 ± 0.10
3.40 ± 0.41 1.52 ± 0.30
0.06 ± 0.01
4.36 ± 0.73 2.24 ± 0.48
1.17 ± 0.23 0.68 ± 0.11
0.89 ± 0.10
1.18 ± 0.29
1.24 ± 0.32
1.33 ± 0.22
1.05 ± 0.15
1.11 ± 0.21
0.71 ± 0.15
0.81 ± 0.19
0.44 ± 0.07
0.78 ± 0.10
4.48 ± 0.98
0.12 ± 0.01
5.51 ± 1.13
1.79 ± 0.44
The in vitro serum stability was evaluated by the incubation of
.7 MBq of Al F–NOTA–BZA in 1 mL of PBS and human serum at
7 °C. The radiochemical purities of Al F–NOTA–BZA in PBS and
human serum were both still >95% after 120 min of incubation as
1
8
3
18
3
L.Int
0.57 ± 0.11
determined by the chromatographic method (ITLC-SG plate, devel-
Spleen
Pancreas
Muscle
Bone
Tumor
Brain
Kidney
Eyeball
Urine
Feces
oping agent 0.05 M Na
bility in vitro.
2 3
CO ), and exhibited a very good serum sta-
0.25 ± 0.04
To assess the binding affinity of Al18F–NOTA–BZA to synthetic
melanin, 0.74 MBq of this radiotracer was added into an aqueous
melanin suspension (0.5 mg/10 mL). The mixture was vortexed
intermittently for 30, 60, 90 and 120 min at 37 °C. The binding of
0.09 ± 0.01
1
8
Al F–NOTA–BZA to melanin was rapid initially (72% after a 30-
min incubation), plateaued at 60 min of incubation (90%), and
remained high till 120 min of incubation (93%, Fig. 3A). In another
study, the binding of Al18F–NOTA–BZA to various concentrations of
340.4 ± 55.9 175.1 ± 38.9 101.6 ± 24.5 39.1 ± 13.5
0.53 ± 0.17
0.61 ± 0.20
0.58 ± 0.17 0.53 ± 0.16
Uptake ratio
Tumor-to-muscle
Tumor-to-blood
Tumor-to-liver
7.70 ± 1.15
1.76 ± 0.49
3.45 ± 0.76
10.2 ± 1.72
3.75 ± 0.69
3.65 ± 0.47
13.8 ± 2.07 16.4 ± 2.55
4.78 ± 0.83 5.96 ± 0.81
4.35 ± 0.79 5.34 ± 0.61
1
8
melanin suspension was determined. About 0.74 MBq of Al F–
NOTA–BZA was mixed with 0.125, 0.25, 0.5, 1 and 2 mg/10 mL of
synthetic melanin suspension and vortexed intermittently for
18
6
0 min at 37 °C. The bound ratio of Al F–NOTA–BZA was almost
the same for the melanin concentration, which ranged from
.125 mg/10 mL (88%) to 2 mg/10 mL (92%) after 60 min of incuba-
tion (Fig. 3B). Our studies show that the binding of Al F–NOTA–
BZA to melanin is dependent on both the melanin concentration
Table 3
Biodistribution of Al18F–NOTA–BZA in BALB/c nude mice bearing A375 tumor after iv
injection of 3.7 MBq Al F–NOTA–BZA. Values were presented as% ID/g (mean ± SD,
0
18
18
n = 3 at each time point)
Organ
0.25 h
0.5 h
1.15 ± 0.32
1 h
2 h
and the incubation time.
In vitro cellular uptake studies of Al18F–NOTA–BZA in B16F0
Blood
Heart
Lung
Liver
Stomach
S.int
3.10 ± 0.84
0.83 ± 0.12
0.85 ± 0.15
1.34 ± 0.35
1.00 ± 0.22
0.75 ± 0.11
0.78 ± 0.13
0.77 ± 0.10
0.70 ± 0.11
0.79 ± 0.25
0.76 ± 0.15
0.87 ± 0.21
0.11 ± 0.02
6.91 ± 1.57
0.68 ± 0.14
198.6 ± 71.4
0.43 ± 0.12
0.45 ± 0.12
0.34 ± 0.11
0.30 ± 0.08
0.52 ± 0.19
0.27 ± 0.09
0.43 ± 0.12
0.39 ± 0.09
0.31 ± 0.09
0.26 ± 0.08
0.28 ± 0.08
0.48 ± 0.13
0.37 ± 0.11
0.07 ± 0.01
2.28 ± 0.65
0.34 ± 0.09
71.3 ± 16.8
0.73 ± 0.14
0.21 ± 0.06
0.13 ± 0.04
0.19 ± 0.07
0.31 ± 0.09
0.16 ± 0.06
0.22 ± 0.07
0.26 ± 0.09
0.24 ± 0.07
0.19 ± 0.06
0.16 ± 0.03
0.23 ± 0.08
0.24 ± 0.09
0.05 ± 0.02
1.34 ± 0.36
0.21 ± 0.08
21.7 ± 6.74
0.92 ± 0.22
0.75 ± 0.15
0.61 ± 0.11
0.81 ± 0.22
0.65 ± 0.18
0.64 ± 0.10
0.72 ± 0.11
0.66 ± 0.12
0.49 ± 0.10
0.42 ± 0.10
0.53 ± 0.11
0.55 ± 0.17
0.09 ± 0.01
5.08 ± 1.26
0.51 ± 0.12
115.8 ± 28.0
0.66 ± 0.15
melanoma cells and A375 amelanotic melanoma cells were con-
ducted. The B16F0 cell line is high melanin expressing, while the
1
8
A375 cell line is non melanin expressing. An aliquot of Al F–
NOTA–BZA was added into each well and incubated at 37 °C for
various time periods (15, 30, 60 and 120 min). A rapid and appre-
L.int
Spleen
Pancreas
Muscle
Bone
Tumor
Brain
Kidney
Eyeball
Urine
Feces
6
ciable uptake in B16F0 melanoma cells (1.13 ± 0.06% AD/10 cells
after 15 min of incubation, Fig. 4) was observed, followed by a
6
gradual increase till 120 min of incubation (1.64 ± 0.06% AD/10
cells, Fig. 4). Although the high hydrophilicity did not appreciably
interfere with the binding affinity of Al18F–NOTA–BZA to melanin,
it may retard the transportation of this tracer through the cell
membrane via diffusion and thus reduce the in vitro cellular
uptake compared with those radiohalogenated probes with lower
hydrophilicity reported in our previous studies.1
6,19
The uptake in
Uptake ratio
Tumor-to-muscle
Tumor-to-blood
Tumor-to-liver
1.17 ± 0.35
0.28 ± 0.10
0.69 ± 0.26
1.28 ± 0.28
0.48 ± 0.17
0.70 ± 0.29
1.32 ± 0.29
0.69 ± 0.13
0.72 ± 0.15
1.50 ± 0.30
1.16 ± 0.23
0.77 ± 0.10
amelanotic A375 melanoma cells, as expected, was very low
6
6
(
0.24 ± 0.02% AD/10 cells at 15 min, 0.18 ± 0.02% AD/10 cells at
1
20 min of incubation). A 9-fold higher uptake of Al18F–NOTA–
BZA in melanotic B16F0 cells than that in amelanotic A375 cells
1
8
after 120 min of incubation indicated that Al F–NOTA–BZA uptake
is associated with melanin.
The biodistribution of Al18F–NOTA–BZA was performed in
C57BL/6 mice bearing B16F0 murine melanoma and in BALB/c
nude mice bearing A375 human amelanotic melanoma. After intra-
1
8
venous injection of Al F–NOTA–BZA into B16F0 melanoma mice, a
rapid and high uptake in high-melanin-expressing B16F0 mela-
noma (6.67 ± 0.91% ID/g at 15 min p.i.) was observed, accompanied
by a slower clearance (1.50 ± 0.26% ID/g at 120 min p.i.) compared
with most normal tissues (Table 2). The relatively sustained
radioactivity retention in tumor rendered favorable tumor-to-nor-
mal tissue ratios for tumor detection. The tumor-to-muscle and
tumor-to-liver ratios (7.70, 3.45 and 16.40, 5.34 at 15 and
120 min p.i., respectively) kept increasing with time. In addition,
the bony uptake was low in both mouse models, indicating that
Figure 4. In vitro cellular uptake of Al18F–NOTA–BZA in B16F0 and A375 cells. Data
are expressed as mean ± SD, with each data point representing triplicate study.