T. Hadizad et al. / Bioorg. Med. Chem. 17 (2009) 7971–7977
7977
[
11C]Methyl iodide was distilled through a P2O5 trap to the reaction
vial with helium by a flow rate of 30 mL/min. Once maximum
11C]methyl iodide is trapped, the reaction vial was sealed and heated
in the time course studies. Data were expressed as a ratio of the
%ID/g of tissue to that of blood to normalize for any differences
in tracer delivery between treatment groups. Group means were
compared using one-way ANOVA and Bonferroni post-hoc analysis
with p <0.05 as significant.
[
at65–70 °C for3 min. The reaction mixture wasthen cooled toꢀ20 °C
and 1 N HCl (0.2 mL) was added. The reaction temperature was raised
to 65–70 °C and heated at this temperature for 2 min. The reaction
mixture was then cooled to ꢀ40 °C, quenched with HPLC buffer (ace-
tonitrile/0.1 M ammonium formate solution = 35/65, 0.3 mL) and in-
Acknowledgements
jected into the semi-preparative HPLC column (Luna C18, 10
l
,
We thank Jeffrey Collins and Paul Coletta for assistance in radio-
chemistry, Stephanie Thorn, Miran Kenk and James Thackeray for
biodistribution studies. We are also grateful to the Canadian Insti-
tutes of Health Research MOP-80203, and the Heart and Stroke
Foundation Molecular Function and Imaging program Grant #
6242 for funding this work.
250 ꢁ 10 mm, 8 mL/min). [11C]Methyl-candesartan was eluted at
8.5 min. The fraction containing the product was collected in a rotary
evaporator,evaporatedtodrynessandthefinalproductwasdissolved
in a saline/sterile water/8.4% sodium bicarbonate (5/4.4/0.66 v/v/v)
mixture. Quality control analyses were determined by injection of a
formulation sample onto Luna C18 (250 ꢁ 4.6 mm, 2 mL/min) using
the same solvent as for semi-preparative HPLC.
References and notes
5.1.6. [11C]-Methyl-1-[[20-(1H-tetrazol-5-yl)biphenyl-4-yl]meth-
yl]-2,3-dihydro-2-oxo-1H-benzimidazole-7-carboxylate
([11C]TH4)
1. Ardaillou, R. J. Am. Soc. Nephrol. 1999, 10, S30.
2. Harada, K.; Sugaya, T.; Murakami, K.; Yazaki, Y.; Komuro, I. Circulation 1999,
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3. Yousef, Z.; Redwood, S.; Marber, M. Heart 2000, 83, 76.
4. Wolf, G.; Ziyadeh, F. Am. J. Kidney Dis. 1997, 29, 153.
5. Admiraal, P.; Danser, A.; Jong, M.; Pieterman, H.; Derkx, F.; Schalekamp, M.
Hypertension 1993, 21, 173.
[
11C]TH4 was obtained using similar procedure as outlined for
[
11C]methyl-candesartan at 90 °C in both the methylation and
deprotection steps. [11C]TH4 was eluted at 4.2 min in semi-pre-
parative HPLC.
6. Opie, L. H.; Sack, M. Circ. Res. 2001, 88, 654.
7. Phillips, M. I.; Speakman, E. A.; Kimura, B. Regul. Pept. 1993, 43, 1.
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5.1.7. Ex vivo bio-testing
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5.1.7.1. Time course. Radiotracer retention time course was inves-
tigated using published methods for C-11 radioligands.29,30 In brief,
male Sprague-Dawley rats (200–275 g, Charles River, Montreal,
Canada) were intravenously injected with 55.5–62.9 MBq (1.5–
1.7 mCi) of [11C]methyl-candesartan or [11C]TH4 at a specific activ-
ity of 11.5–37 GBq/lmol of the respective tracer (311–1000 mCi/
lmol at time of first injection; 2.4–10.8
lg/kg mass injected). Rats
were sacrificed by decapitation without anesthesia 5, 15, 30, 45 or
60 min after [11C]methyl-candesartan injection, and 5, 15, 30, or
60 min after [11C]TH4 injection. Tissues were quickly dissected
out and collected into pre-weighed gamma tubes: heart, kidney
(renal cortex, outer medulla and inner medulla separately), adre-
nal, brain, lung, liver, and skeletal muscle (quadriceps). Trunk
blood was collected in heparinized tubes, from which 1 mL was re-
moved and transferred to pre-weighed gamma tubes. Blood and
tissues were counted (decay-corrected) in a gamma-counter (Pack-
ard) along with injected standard solutions. Data were expressed
as percent of injected dose per gram of tissue (%ID/g).
21. Zober, T. G.; Mathews, W. B.; Seckin, E.; Yoo, S. E.; Hilton, J.; Xia, J.; Sandberg,
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T.; Nishikawa, K.; Naka, T. J. Med. Chem. 1993, 36, 2343.
25. Naka, T.; Nishikawa, K.; Kato, T. U.S. Patent 5,196,444, 1993.
26. Duncia, J. V.; Pierce, M. E.; Santella, J. B., III J. Org. Chem. 1991, 56, 2395.
27. Chang, R. S.; Lotti, V. J. Life Sci. 1991, 49, 1485.
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Walther, S.; Sexton, P. M.; Gembardt, F.; Kellett, E.; Martini, L.; Vanderheyden,
P.; Schultheiss, H. P.; Walther, T. Circulation 2005, 111, 1806.
29. Lourenco, C. M.; Houle, S.; Wilson, A. A.; DaSilva, J. N. Nucl. Med. Biol. 2001, 28,
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30. Kenk, M.; Greene, M.; Thackeray, J.; Dekemp, R. A.; Lortie, M.; Thorn, S.;
Beanlands, R. S.; Dasilva, J. N. Nucl. Med. Biol. 2007, 34, 71.
31. Nossaman, B. D.; Baber, S. R.; Nazim, M. M.; Detrolio, J. D.; Kadowitz, P. J. Can. J.
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Yip, H. K. Int. Heart J. 2007, 48, 533.
33. Naruse, M.; Tanabe, A.; Sato, A.; Takagi, S.; Tsuchiya, K.; Imaki, T.; Takano, K.
Hypertension 2002, 40, 28.
34. Bayorh, M. A.; Eatman, D.; Walton, M.; Socci, R. R.; Thierry-Palmer, M.; Emmett,
N. Peptides 2002, 23, 57.
35. Friedman, E. M.; Irwin, M. Brain Behav. Immun. 2001, 15, 65.
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5.1.7.2. Competition. Radiotracers were evaluated for in vivo bind-
ing selectivity for the AT1 receptor subtype using the published pro-
cedures29,30 described above. AT1 receptor blockers candesartan (3,
5 or 10 mg/kg)31 or losartan (20 or 30 mg/kg)32 were co-adminis-
tered iv with the tracer or intravenously injected 5 min prior to radi-
oligand injection. The AT2 antagonist PD123,319 (2 or 5 mg/kg for
[
11C]methyl-candesartan; 5 mg/kg for [11C]TH4),21,33 and a Mas
receptor (Ang 1–7) antagonist A-779 (100
g/kg)34 were injected
l
through the tail vein, 5 min prior to radioligand injection. Interperi-
toneal injections of the b-adrenergic receptor blocker propranolol
(20 mg/kg)35 or the
a2-adrenergic receptor blocker yohimbine
(10 mg/kg)36 were given 15 min prior to the radioligand tail vein
injection.
Rats (200–275 g) were sacrificed by decapitation without anes-
thesia 15 min following radioligand injection. Trunk blood and tis-
sue samples or whole organ samples were collected and counted as