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T. Chu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 658–661
Table 1
Uptake of [131I]2NPBTA8 and [131I]2NUBTA in the gerbil ischemic and normal brain hemisphere
Brain
[
131I]2NPBTA8
[
131I]2NUBTA
4 h
8 h
12 h
4 h
8 h
12 h
Right
Left
Right/left
0.042 0.005
0.036 0.004
1.18 0.13
0.034 0.006
0.025 0.003
1.39 0.10
0.025 0.004
0.014 0.002
1.76 0.10
0.233 0.021
0.151 0.029
1.57 0.29
0.174 0.015
0.097 0.017
1.82 0.28
0.127 0.008
0.049 0.012
2.76 0.96
Each value is mean standard deviation.
further partition coefficient determination and in vitro and in vivo
study. Being kept at room temperature for one week, the radio-
chemical purity of the product was still >95%. Five milliliters of
1-octanol, 5 mL of water, and 0.1 mL of radioiodinated product
were mixed by vigorously vortexing at room temperature for
5 min. Then it was centrifuged at 4000 rpm for 5 min, and the
phases were separated. Sample (0.1 mL) was taken from each
phase and counted. The octanol/water partition coefficient (P)
was the ratio of the radioactivity of octanol phase to that of aque-
ous phase. Then the water layer was removed and the same vol-
ume of fresh water was added until consistent P values were
obtained. This measurement was repeated for three times. The
logP of [131I]2NUBTA was 1.99, higher than that of previously re-
ported cerebral ischemia marker [131I]2NPBTA (logP = 1.56).7,8
Obviously, by adding the length of the linker between 2-nitroimi-
dazole and BTA, the lipophilicity of new cerebral ischemia marker
was incomplete. Therefore, the blood supply of each hemisphere
was isolated from the contralateral carotid and basilar arteries.
Ligation of one carotid artery caused ischemia in the ipsilateral
hemisphere, while the other side was unaffected, providing neigh-
boring normal tissue as an internal control. The unique anatomical
feature of the gerbil made them widely used as a model in global
ischemia.
Adult mongolian gerbils (male, 80 g) were used for stroke mod-
els. They had been subjected to right common carotid artery liga-
tion to produce cerebral hypoxia–ischemia (HI) as initially
described by Levine and Payan9 and the stroke index described
by Ohno et al.10 was calculated. Animals with total stroke indices
of >10 were used for injection.
[
131I]2NUBTA (0.5 mL,
1.0 Â 105 Bq) was injected intraperitoneally (ip) into the gerbils.
At the time of sacrifice, animals with total stroke indices of >10
were killed (no anesthesia) by cervical dislocation in groups of
three at 4, 8, and 12 h after injection. The whole brain was re-
moved, placed on dry ice for 2 min, and then cut in half along
the cerebral longitudinal fissure. The right and left halves were
weighed and radioactivity counted. The percent injected dose per
gram of tissue, that is, % ID/g, was determined for the right and left
hemispheres. The right/left hemispheral uptake ratios were calcu-
lated. All experiments were carried out following the principles of
laboratory animal care and the China law on the protection of
animals.
[
131I]2NUBTA increased.
Cellular accumulation. S180 cells were suspended in 20 mL Dul-
becco’s modified Eagle’s medium (DMEM) containing 10% (v/v) of
fetal bovine serum at a concentration of (1–2) Â 106 cells/mL and
incubated at 37 °C. The hypoxic and aerobic conditions were gen-
erated by stirring the cell suspension under an atmosphere of
nitrogen or air, respectively (both containing 5% CO2). In the hyp-
oxic conditions, after pre-equilibrated for 40 min, the oxygen sen-
sor of the Dissolved Oxygen Meter read 0.00–0.01 mg/L. In the
aerobic conditions, no pre-equilibration was performed, the oxy-
The final results were expressed as means standard deviation
(SD) as listed in Table 1. The uptake of [131I]2NUBTA in the right
hemisphere was higher than that in the left at 4, 8, and 12 h
post-injection. The results indicated that the clearance from ische-
mic brain tissue was slower than that from normal brain tissue.
The right/left uptake ratios, that is, the uptake ratios of ischemic
to normal brain tissues were gradually increasing for 2NUBTA,
from 1.57 at 2 h to 2.76 at 12 h. The difference between the uptake
of the right hemisphere and the left hemisphere was very signifi-
cant (P value <0.01) at 8 and 12 h.
gen meter read ꢀ6.00 mg/L.
[
131I]2NUBTA (0.2 mL, 148 kBq,
L, in
13.5 nmol) was added to the suspension and aliquots (200
l
pentaplicate) were pipetted using a syringe through rubber plug
to keep the oxygen out of the cell suspension at 5, 30, 60, 90,
120, 150 min, and centrifuged at 1500 rpm for 5 min. A 90
ple of each supernatant was removed for counting (A), each tube
containing cells and 110 L medium with resuspending before
counting was also counted (B). The accumulation ratio, Cin/Cout
lL sam-
l
,
was defined as concentration of radioactivity within the cells di-
vided by that in the external medium, and calculated as [(B À A)/
And the results of the uptake of [131I]2NPBTA, with lower lipo-
A], because the volume of cells in 200 lL of cell suspension is about
philicity (logP = 1.56) reported previously8 were also listed in
20
l
L. The final results were expressed as means standard devia-
Table 1 for comparison. It was found that the uptakes of
tion (SD) of five independent parallel experiments. During the
whole procedure, the cells maintained >90% viability.
[
131I]2NUBTA (logP = 1.99) in both the right hemisphere and the
left hemisphere were higher than those of [131I]2NPBTA, and espe-
cially the right/left uptake ratio of [131I]2NUBTA was also higher
than that of [131I]2NPBTA, such as 2.76 for [131I]2NUBTA, 1.76 for
The Cin/Cout ratios under hypoxic and aerobic conditions were
plotted as a function of time as showed in Figure 3. The accumula-
tion of [131I]2NUBTA steadily increased with time in hypoxic cells,
but fluctuated with time and had no fixed trend in aerobic cells.
The Cin/Cout values at 5 min for hypoxic and aerobic cells were
0.52 and 0.31, respectively, for [131I]2NUBTA, while at 4 h the Cin/
Cout was 0.74 for hypoxic cells and 0.29 for aerobic cells, represent-
ing a 2.6-fold hypoxic/aerobic difference. At 4 h, the difference be-
tween hypoxic cells and aerobic cells was very significant and the P
values (two-tailed) <0.01 (P value was determined using Student’s
t-test). The results indicated [131I]2NUBTA could retain in hypoxic/
ischemic tissue but flow out from normal tissue with time going,
when it was injected intraperitoneally (ip) into animals.
[
131I]2NPBTA at 12 h post-injection, respectively.
Therefore, after adding the length of the linker between 2-nitro-
imidazole and BTA to increase the lipophilicity, [131I]2NUBTA may
be a more suitable cerebral ischemia marker than [131I]2NPBTA.
Acknowledgments
We thank the National Natural Science Foundation of China
(Grant No. 20771011) and the Ministry of Science and Technology
of China (Grant No. 2006CB705700) for financial support. The
assistance of Dr. Weihong Cong and Prof. Jianxun Liu, Xiyuan Hos-
pital, China Academy of Traditional Chinese Medicine in the
in vitro and in vivo studies is gratefully acknowledged.
Evaluation in gerbil cerebral ischemia models. The gerbils did not
have a posterior communicating artery, that is, the circle of Willis