- Email: [email protected]
BCRP knockout mice
Accepted Manuscript [11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice Yoko Shimoda, Joji Yui, Masayuki Fujinaga, Lin Xie, Katsushi Kumata, Masanao Ogawa, Tomoteru Yamasaki, Akiko Hatori, Kazunori Kawamura, Ming-Rong Zhang PII: DOI: Reference:
S0960-894X(14)00545-9 http://dx.doi.org/10.1016/j.bmcl.2014.05.045 BMCL 21659
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
31 March 2014 2 May 2014 14 May 2014
Please cite this article as: Shimoda, Y., Yui, J., Fujinaga, M., Xie, L., Kumata, K., Ogawa, M., Yamasaki, T., Hatori, A., Kawamura, K., Zhang, M-R., [11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/ 10.1016/j.bmcl.2014.05.045
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[11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice
Yoko Shimodaa, Joji Yuia, Masayuki Fujinagaa, Lin Xiea, Katsushi Kumataa, Masanao Ogawaa,b, Tomoteru Yamasakia, Akiko Hatoria, Kazunori Kawamuraa, and Ming-Rong Zhanga*
a)
Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological
Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan; b)SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
*
Address correspondence to: Ming-Rong Zhang, Molecular Probe Program, Molecular
Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, Tel.: 81-43-382-3709, Fax: 81-43-206-3261, E-mail: [email protected]
Keywords: [11C]CEP-32496, PET, BRAF, P-gp, BCRP, [11C]phosgene
1
Abstract CEP-32496 is a novel, orally active serine/threonine-protein kinase B-raf (BRAF) (V600E) kinase inhibitor that is being investigated in clinical trials for the treatment of some cancers in patients. In this study, we developed [11C-carbonyl]CEP-32496 as a novel positron emission tomography (PET) probe to study its biodistribution in the whole bodies of mice. [11C]CEP-32496
was
synthesized
by
5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine
the
reaction
hydrochloride
(1•HCl)
of with
[11C]phosgene, followed by treatment with 3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2). Small-animal PET studies with [11C]CEP-32496 indicated that radioactivity levels (AUC0-90 min,
SUV × min) accumulated in the brains of P-gp/BCRP knockout mice at a 8-fold higher
rate than in the brains of wild-type mice.
2
The V600E driver mutations in the serine/threonine-protein kinase B-raf (BRAF) kinase gene has been confirmed in approximately 7% of all cancers, including 60–70% of melanomas, 29–83% of papillary thyroid carcinomas, 4–16% colorectal cancers, and to a lesser degree in non-small cell lung carcinomas and ovarian cancers.1 The V600E mutation is an activating process and was found to
cause
diseases,
including
melanoma,
thyroid,
colon,
lung
and
ovarian cancers.2
1-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol -3-yl)urea hydrochloride (CEP-32496; Scheme 1) exhibits high in vitro inhibitory affinity (Kd = 14 nM) for BRAF mutant.3 Preclinical studies have shown that this drug has high antitumor ability in xenograft models of melanoma and colon cancer.3,4 Thus, the orally active CEP-32496 as a molecular target drug may enable targeted therapy in patients with BRAF driver mutations, and is currently being evaluated for efficacy in clinical trials.
Scheme 1
Many molecular target drugs, such as gefitinib, imatinib and sorafenib, are substrates for P-glycoprotein (P-gp, Abcb1a/1b) and breast cancer resistance protein (BCRP, ABCG2), two representative ATP-binding cassette (ABC) transporters.5-7 ABC transporters can actively transport internalized drugs across cell membranes into extracellular regions; thus, their overexpression in tumor cells may result in drug resistance. Numerous studies have shown that P-gp limits the adsorption and distribution of anticancer drugs in the brain and in tumor cells,8-10 which may inhibit their efficacy in combating tumor growth in the brain and other organs. It is therefore important to evaluate the influence of P-gp and BCRP on the pharmacokinetics of anticancer drugs. However, despite clinical trials of CEP-32496 in patients, to our knowledge the potential contribution of P-gp and/or BCRP to resistance against CEP-32496 has not been reported.
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As a molecular imaging modality, positron emission tomography (PET) enables the study of drug pharmacokinetics and pharmacodynamics, and is thus important for both drug discovery and drug development.11 Using radioactive probes in PET aids the study of P-gp/BCRP functions at the blood-brain-barrier (BBB), as well as the evaluation of therapeutic effects of drugs in combating various diseases.12,13 Previously, we have developed the PET probes [11C]gefitinib14,15 and [11C]sorafenib,16 and showed that the pharmacokinetics of both molecular target drugs were significantly affected by both P-gp and BCRP. The aims of this study were to develop [11C]CEP-32496 as a novel PET probe to determine its biodistribution profile in mice and to characterize the influence of P-gp/BCRP-mediated efflux on the uptake of [11C]CEP-32496 in mouse brains. To achieve the radiosynthesis of [11C]CEP-32496, we utilized a reliable method for constructing an unsymmetrical [11C-carbonyl]urea moiety by the reaction of [11C]phosgene ([11C]COCl2)17-19 with two different amines. This approach involves the reaction of an amine hydrochloride with [11C]COCl2, followed by treatment with another free amine to produce the desired unsymmetrical [11C]urea.16,20-22 Here, we applied this technique to synthesize [11C-carbonyl]CEP-32496
(Scheme
1).
CEP-32496
5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine
and
two (1)
precursors: and
3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2), were prepared in house following reported methods,3 with minor modification. The identities of these compounds were confirmed by NMR and high-resolution mass spectra (HRMS).23 After optimizing the reaction conditions, such as reaction temperatures, reaction times, and the amounts of amines 1 and 2 used, we automated the radiosynthesis of [11C]CEP-3249624 with a synthesis system18,19 developed in house. Figure 1A shows a representative HPLC chromatogram for the separation, in which [11C]CEP-32496 was produced as the main radioactive peak in the
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reaction mixture. This protocol yielded 0.76–1.11 GBq of [11C]CEP-32496 at the end of synthesis (EOS), starting with 22–23 GBq of [11C]CO2 (n = 5). The averaged radiochemical yield of [11C]CEP-32496 was 14 ± 3% based on [11C]CO2 (decay-corrected at the end of bombardment (EOB)). The total synthesis time was 35 ± 3 min from EOB.
Figure 1
The identity of [11C]CEP-32496 was confirmed by co-injection with authentic CEP-32496 using analytic HPLC.24 The radiochemical purity of the final [11C]CEP-32496 product in solution was >98% (Figure 1B) and its specific activity was 34–72 GBq/µmol (n = 5) at EOS. No significant UV peaks corresponding to unlabeled 1 and 2 were observed on the HPLC chart. Moreover, the radiochemical purity remained at ~98% after storage at room temperature for 90 min, indicating that [11C]CEP-32496 was radiochemically stable for the duration of at least one PET scan. These analytical results complied with our in-house quality control/assurance specifications.
Table 1
We first studied the biodistribution of [11C]CEP-32496 in the whole bodies of mice (n = 4). Table 1 shows the distribution of radioactivity in mouse tissues following i.v. injection of a saline solution of [11C]CEP-32496 (8.4 MBq/0.1 mL). Radioactivities in tissues are expressed as the percentage of injected dose per gram of wet tissue (% ID/g). High initial radioactivity levels (>10% ID/g) were found in the heart, lung, liver and kidney at 1 min after injection. Radioactivity levels in the blood, heart and lung declined rapidly by 5 min and then decreased slowly over the measurement period of 60 min. The uptake of [11C]CEP-32496 in the liver and pancreas was
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maintained at relatively consistent levels throughout this experiment. Radioactivity gradually accumulated in the small intestine and reached a maximum of 25.4% ID/g at 30 min. These results suggest that the hepatobiliary, urinary excretion and intestinal reuptake pathway dominated the whole-body distribution of [11C]CEP-32496. Compared to peripheral tissues, brain tissues showed low levels of radioactivity uptake (0.21–0.08% ID/g), indicating that [11C]CEP-32496 could not enter the brain easily.
Figure 2
We then carried out a PET study with [11C]CEP-32496 using wild-type and P-gp/BCRP knockout mice under anesthesia with 1–2% isoflurane to compare their relative levels of [11C]CEP-32496 uptake in the brain, which was expressed as the standardized uptake value (SUV).25 Figure 2 shows representative coronal and horizontal PET brain images from wild-type (A, B) and P-gp/BCRP knockout mice (C, D). The radioactivity accumulated in the brains of wild-type mice was markedly lower than that observed with P-gp/BCRP knockout mice. Figure 2E shows time-activity curves in whole brains from both experimental groups. Brains from P-gp/BCRP knockout mice retained high levels of radioactivity, which reached a stable plateau in PET scans between 20 and 60 min after injection of [11C]CEP-32496. Radioactivity levels, measured as areas under the time-activity curves AUC0-90 min (SUV × min) between 0 and 90 min after injection of [11C]CEP-32496, were 49.34 ± 1.5 (n = 3) and 6.1 ± 0.4 (n = 3) for P-gp/BCRP knockout mice and wild-type mice, respectively. The ratio of AUC0-90
min
in the
two-type mice was 8:1. Differences in AUC0-90 min values between the two groups were statistically significant (P < 0.05, Student’s paired t-test).
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Figure 3
To confirm the results of PET study, we harvested blood and dissected brains from P-gp/BCRP knockout and wild-type mice (n = 5 for each group) to determine radioactivity levels (% ID/g) at 60 min after injection of [11C]CEP-32496. As shown in Figure 3, radioactivity uptake in brains from the knockout mice was 24-fold higher than observed with wild-type mice. However, no significant difference was observed in blood uptake levels between the two groups. This study indicates that the uptake of [11C]CEP-32496 in mouse brains may be limited by P-gp and/or BCRP at the BBB. Our results demonstrate for the first time that CEP-32496, like gefitinib and sorafenib, may be a substrate for P-gp and/or BCRP. [11C]CEP-32496 is a promising PET probe that may be used to evaluate and characterize P-gp and/or BCRP functions in vivo. As CEP-32496 is a candidate anticancer drug, PET studies with [11C]CEP-32496 may be useful for BRAF kinase imaging in tumors and for evaluating its bioavailability in combination with other chemotherapeutics against tumors including brain tumors. For example, PET studies may be useful for evaluating the effects of dual P-gp and BCRP inhibitors, such as elacridar,26,27 on the brain permeation with [11C]CEP-32496, in attempt to improve the therapeutic efficacy of CEP-32496 in treating patients with brain tumors. In conclusion, CEP-32496 was successfully labeled with
11
C on its urea group using
[11C]COCl2. [11C-carbonyl]CEP-32496 was produced with sufficiently high specific radioactivity and purity to be acceptable for animal studies. Further studies regarding the effects of P-gp or/and BCRP on the penetration of [11C]CEP-32496 into brain tumors and BRAF kinase imaging in animal models bearing tumors are in progress.
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Acknowledgements We are grateful to Mrs. Y. Kurihara and N. Nobuki (SHI Accelerator Service Co. Ltd.) for providing technical support with radiosynthesis and to Mr. H. Wakizaka (National Institute of Radiological Sciences) for providing assistance with PET scans. We also thank the staff of the National Institute of Radiological Sciences for support in operating the cyclotron operation and the production of radionuclide.
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N.; Dobrzanski, P.; Gardner, M. F.; Zhao, H.; Cramer, M. D.; Hunter, K.; Nepomuceno, R. R.; Cheng, M.; Gitnick, D.; Yazdanian, M.; Insko, D. E.; Ator, M. A.; Apuy, J. L.; Faraoni, R.; Dorsey, B. D.; Williams, M.; Bhagwat, S. S.; Holladay, M. W. Mol. Cancer Ther. 2012, 11, 930. 5. Carcaboso, A. M.; Elmeliegy, M. A., Shen, J.; Juel, S. J.; Zhang, Z. M.; Calabrese, C.; Tracey, L.; Waters, C. M.; Stewart, C. F. Cancer Res. 2010, 70, 4499. 6. Oostendorp, R. L.; Marchetti, S.; Beijnen, J. H.; Mazzanti, R.; Schellens, J. H. Cancer Chemother. Pharmacol. 2007, 59, 855. 7. Harmsen, S.; Meijerman, I.; Maas-Bakker, R. F.; Beijnen, J. H.; Schellens, J. H. Eur J Pharm Sci. 2013, 48, 644. 8. Gottesman, M. M.; Pastan I. Annu. Rev. Biochem. 1993, 62, 385. 9. Schinkel, A. H.; Wagenaar, E.; van Deemter, L.; Mol, C. A.; Borst, P. J. Clin. Invest. 1995, 96, 1698. 10. Dai, H.; Marbach, P.; Lemaire, M.; Hayes, M.; Elmquist, W. F. J. Pharmcol. Exp. Ther. 2003, 304, 1085. 11. Murphy, P. S.; Bergström, M. Curr. Pharm. Des. 2009, 15, 957 12. Mairinger, S.; Erker, T.; Muller, M.; Langer, O. Curr. Drug Metab. 2011, 12, 774. 13. Yamasaki, T.; Kawamura, K.; Hatori, A.; Yui, J.; Yanamoto, K.; Yoshida, Y.; Ogawa, M.; Nengaki, N.; Wakisaka, H.; Fukumura, T.; Zhang, M.-R. Nucl. Med. Commun. 2010, 31, 985. 14. Kawamura, K.; Yamasaki, T.; Yui, J.; Hatori, A.; Konno, F.; Kumata, K.; Konno, F.; Irie, T.; Fukumura, T.; Suzuki, K.; Kanno, I.; Zhang M.-R. Nucl. Med. Biol. 2009, 36, 239. 15. Zhang, M.-R.; Kumata, K.; Hatori, A.; Takai, N.; Toyohara, J.; Yamasaki, T.; Yanamoto, K.; Yui, J.; Kawamura, K.; Koike, S.; Ando, K.; Suzuki, K. Mol. Imaging. Biol. 2010, 12, 181. 16. Asakawa, C.; Ogawa, M.; Kumata, K.; Fujinaga, M.; Kato, K.; Yamasaki, T.; Yui, J.; Kawamura, K.; Hatori, A.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2011, 21, 2220.
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17. Roeda, D.; Dollé, F. Curr. Top. Med. Chem. 2010, 10, 1680. 18. Ogawa, M.; Takada Y., Suzuki H., Nemoto K., Fukumura T., Nucl. Med. Biol. 2010, 37, 73. 19. Takada, Y.; Ogawa, M.; Suzuki, H.; Nemoto, K.; Fukumura, T. Appl. Radiat. Isot. 2010, 68, 1715. 20. Conway, T.; Diksic, M. J. Nucl. Med. 1988, 29, 1957. 21. Asakawa, C.; Ogawa, M.; Fujinaga, M.; Kumata, K.; Xie, L.; Yamasaki, T.; Yui, J.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2012, 22, 3594. 22. Asakawa, C.; Ogawa, M.; Kumata, K.; Fujinaga, M.; Yamasaki, T.; Xie, L.; Yui, J.; Kawamura, K.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2011, 21, 7017. 23. Measured properties of the free base of CEP-32496: mp: 203–204 °C; 1H NMR (DMSO-d6, 300 MHz): δ 1.53 (s, 6H, CH3), 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.87 (s, 1H, Ar–H), 6.96–7.00 (m, 1H, Ar–H), 7.25–7.28 (m, 1H, Ar–H), 7.38–7.44 (m, 2H, Ar–H), 7.56–7.58 (m, 2H, Ar–H), 8.56 (s, 1H, Ar–H), 9.01 (s, 1H, Ar–H), 9.74 (s, 1H, Ar–H); HRMS (FAB) calcd for C24H23O5N5F3, 518.1651; found, 518.1606. Measured properties of compound 1: mp: 100–101 °C; 1H NMR (CDCl3, 300 MHz): δ 1.52 (s, 6H, CH3), 4.04 (brs, 2H, NH2), 5.79 (s, 1H, Ar–H); HRMS (FAB) calcd for C7H10ON2F3, 195.0745; found, 195.0758. Measured properties of compound 2: mp: 186–187 °C; 1H NMR (DMSO-d6, 300 MHz): δ 3.97 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 5.28 (brs, 2H, NH2), 6.36–6.43 (m, 2H, Ar–H), 6.46–6.50 (m, 1H, Ar–H), 7.05–7.11 (m, 1H, Ar–H), 7.37 (s, 1H, Ar–H), 7.51 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H); HRMS (FAB) calcd for C16H15O3N3, 297.1113; found, 297.1109. 24. Compound 1•HCl was prepared by mixing 1 (0.61 mg, 3.14 µmol) in THF (500 µL) with 4 M HCl in 1,4-dioxane (4 µL), and [11C]COCl2 gas, which was produced from cyclotron-generated [11C]CO2, was bubbled into the solution at −15 °C for 1 min. After the trapping of [11C]COCl2 was finished, 2 (2.72 mg, 9.15 µmol) and N,N-dimethyl-4-aminopyridine (0.41 mg, 3.36 µmol)
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in THF (200 µL) were added and the reaction mixture was heated to 70 °C for 5 min. Following the removal of THF, the reaction mixture was diluted with HPLC solvent (2 mL) and applied to a reversed-phase HPLC system. HPLC was performed using a JASCO HPLC system (JASCO, Tokyo) and Capcell Pak C18 (Shiseido, Tokyo) columns. A 10 mm × 250 mm column was used for purification, and a 4.6 mm × 250 mm was used for analysis. Ultraviolet detection was performed at 254 nm. A solution of CH3CN/H2O (60/40, v/v) was used as the eluent. Purification was performed using a liquid phase flow rate of 5.0 mL/min for 7.7 min, and analysis was performed using a liquid phase rate of 1.0 mL/min for 7.4 min. The radioactive fraction corresponding to the desired product was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed through a 0.22 µm Millipore filter to give [11C]CEP-32496. 25. PET scans were performed using a small-animal Inveon PET scanner (Siemens, Knoxville, TN), which provides 159 transaxial slices with 0.796 mm (center-to-center) spacing, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial FOV. A bolus of 17–19 MBq of [11C]CEP-23496 (0.44 to 1.08 nmol in 100 µL saline) was injected into the tail vein of mice. Wild-type (male, 18–19 weeks old, 29–31 g, n = 3) and P-gp/BCRP knockout (Abcb1a/1b-/-Abcg2-/-, male, 18–19 weeks old, 35–39 g, n = 3) mice (FVB, Taconic Farm, Hudson, NY) were used. A dynamic emission scan was performed in 3D list-mode for 90 min (1 min × 4 frames, 2 min × 8 frames, 5 min × 14 frames). Region of interest (ROI) analyses and image reconstruction were performed using the ASIPro software (Siemens Medical Solutions). Visual analyses were performed by individuals experienced in PET interpretation, using coronal and horizontal reconstructions. ROIs were manually placed across image planes for time-activity curves. Radioactivity was decay-corrected to the injection time and expressed as a standardized uptake value (SUV) normalized for injected radioactivity and body weight, according to the
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formular: SUV = (radioactivity per cm3 tissue/[injected radioactivity × body weight in grams). The area under the time–activity curve of the ROIs in the brain (AUC0-90 min, SUV × min) was calculated from 0 to 90 min. 26. Lagas, J. S.; Waterschoot, R. A.; Tilburg, V. A.; Hillebrand, M. J.; Lankheet, N.; Rosing, H.; Beijnen, J. H.; Schinkel, A. H. Clin. Cancer Res. 2009, 15, 2344. 27. Kawamura, K.; Yamazaki, T.; Konno, F.; Yui, J.; Hatori, A.; Yanamoto, K.; Wakizaka, H.; Fukumura, T.; Zhang, M.-R. Mole. Imaging Biol. 2011, 13, 152.
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Figure Legends
Scheme 1. Chemical structure of CEP-32496 and radiosynthesis of [11C-carbonyl]CEP-32496: (a) THF, room temperature, 5 min, (b) THF, −15 °C, 1 min; (c) N,N-dimethyl-4-aminopyridine, THF, 70 °C, 5 min, 72% (decay-corrected, analyzed by HPLC for the final reaction mixture).
Figure 1. Chromatograms from the HPLC separation (A) and analysis (B) used in the radiosynthesis of [11C-carbonyl]CEP-32496.
Figure 2. PET imaging of brains in wild-type- and P-gp/BCRP knockout mice. Panels A and B are images of wild-type mouse, and panels C and D are images of P-gp/BCRP knockout mouse. Coronal images are shown in panels A and C, and horizontal images are shown in panels B and D. Panel E shows the time-activity curves in the brains of wild-type- and P-gp/BCRP knockout mice. Data are the means ± SEM (n = 3 for each group).
Figure 3. Radioactivity in the brains and blood of wild-type- and P-gp/BCRP knockout mice at 60 min after injection of [11C]CEP-32496. Data are the means ± SEM (n = 5 for each group).
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Scheme 1. Shimoda et al.
(A) [11C]CEP-32496
(B) [11C]CEP-32496
Figure 1. Shimoda et al.
A
B
E 0.7 0.6 0.5
C
SUV
0.4
D
P-gp/BCRP knockout Wild type
0.3
0
Radioactivity (SUV)
1
0.2 0.1 0.0 0
10
20
30
40 50 min
60
70
80
90
Figure 2. Shimoda et al.
1.6 1.4 P-gp/BCRP knockout Wild type
1.2 % ID/g
1.0 0.8 0.6 0.4 0.2 0.0 Brain
Blood
Figure 3. Shimoda et al.
TABLE 1. Biodistribution in mice Tissue 1 min 5 min 15 min 30 min Blood 3.14 ± 0.25 0.99 ± 0.05 0.95 ± 0.08 0.87 ± 0.03 Heart 11.25 ± 0.46 4.43 ± 0.27 3.56 ± 0.24 3.08 ± 0.08 Lung 14.30 ± 1.16 4.75 ± 0.43 3.76 ± 0.17 3.34 ± 0.09 Liver 17.18 ± 1.82 15.30 ± 0.94 17.40 ± 1.47 18.42 ± 0.44 Pancreas 2.96 ± 0.15 2.77 ± 0.33 3.52 ± 0.14 3.94 ± 0.24 Spleen 3.92 ± 0.28 2.05 ± 0.17 2.05 ± 0.14 2.03 ± 0.05 Kidney 12.33 ± 0.40 8.36 ± 0.80 7.68 ± 0.50 7.44 ± 0.21 Small intestine 5.92 ± 0.51 14.31 ± 1.38 22.18 ± 1.30 25.40 ± 2.10 Large intestine 0.81 ± 0.04 0.76 ± 0.10 1.01 ± 0.08 1.69 ± 0.20 Muscle 2.37 ± 0.15 1.52 ± 0.18 1.60 ± 0.14 1.54 ± 0.05 Brain 0.21 ± 0.02 0.10 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 Data are % injected dose/g wet tissue (% ID/g) (mean ± SEM ; n = 4).
60 min 0.61 ± 0.03 1.98 ± 0.07 2.24 ± 0.09 12.69 ± 0.48 2.66 ± 0.08 1.36 ± 0.05 4.91 ± 0.13 21.23 ± 2.18 1.98 ± 0.06 1.06 ± 0.04 0.08 ± 0.00
Table 1. Shimoda et al.
Graphical abstract
[11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice
Yoko Shimodaa, Joji Yuia, Masayuki Fujinagaa, Lin Xiea, Katsushi Kumataa, Masanao Ogawaa,b, Tomoteru Yamasakia, Akiko Hatoria, Kazunori Kawamuraa, and Ming-Rong Zhanga*
a)
Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan; b)SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
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S0960-894X(14)00545-9 http://dx.doi.org/10.1016/j.bmcl.2014.05.045 BMCL 21659
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
31 March 2014 2 May 2014 14 May 2014
Please cite this article as: Shimoda, Y., Yui, J., Fujinaga, M., Xie, L., Kumata, K., Ogawa, M., Yamasaki, T., Hatori, A., Kawamura, K., Zhang, M-R., [11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/ 10.1016/j.bmcl.2014.05.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
[11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice
Yoko Shimodaa, Joji Yuia, Masayuki Fujinagaa, Lin Xiea, Katsushi Kumataa, Masanao Ogawaa,b, Tomoteru Yamasakia, Akiko Hatoria, Kazunori Kawamuraa, and Ming-Rong Zhanga*
a)
Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological
Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan; b)SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
*
Address correspondence to: Ming-Rong Zhang, Molecular Probe Program, Molecular
Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, Tel.: 81-43-382-3709, Fax: 81-43-206-3261, E-mail: [email protected]
Keywords: [11C]CEP-32496, PET, BRAF, P-gp, BCRP, [11C]phosgene
1
Abstract CEP-32496 is a novel, orally active serine/threonine-protein kinase B-raf (BRAF) (V600E) kinase inhibitor that is being investigated in clinical trials for the treatment of some cancers in patients. In this study, we developed [11C-carbonyl]CEP-32496 as a novel positron emission tomography (PET) probe to study its biodistribution in the whole bodies of mice. [11C]CEP-32496
was
synthesized
by
5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine
the
reaction
hydrochloride
(1•HCl)
of with
[11C]phosgene, followed by treatment with 3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2). Small-animal PET studies with [11C]CEP-32496 indicated that radioactivity levels (AUC0-90 min,
SUV × min) accumulated in the brains of P-gp/BCRP knockout mice at a 8-fold higher
rate than in the brains of wild-type mice.
2
The V600E driver mutations in the serine/threonine-protein kinase B-raf (BRAF) kinase gene has been confirmed in approximately 7% of all cancers, including 60–70% of melanomas, 29–83% of papillary thyroid carcinomas, 4–16% colorectal cancers, and to a lesser degree in non-small cell lung carcinomas and ovarian cancers.1 The V600E mutation is an activating process and was found to
cause
diseases,
including
melanoma,
thyroid,
colon,
lung
and
ovarian cancers.2
1-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol -3-yl)urea hydrochloride (CEP-32496; Scheme 1) exhibits high in vitro inhibitory affinity (Kd = 14 nM) for BRAF mutant.3 Preclinical studies have shown that this drug has high antitumor ability in xenograft models of melanoma and colon cancer.3,4 Thus, the orally active CEP-32496 as a molecular target drug may enable targeted therapy in patients with BRAF driver mutations, and is currently being evaluated for efficacy in clinical trials.
Scheme 1
Many molecular target drugs, such as gefitinib, imatinib and sorafenib, are substrates for P-glycoprotein (P-gp, Abcb1a/1b) and breast cancer resistance protein (BCRP, ABCG2), two representative ATP-binding cassette (ABC) transporters.5-7 ABC transporters can actively transport internalized drugs across cell membranes into extracellular regions; thus, their overexpression in tumor cells may result in drug resistance. Numerous studies have shown that P-gp limits the adsorption and distribution of anticancer drugs in the brain and in tumor cells,8-10 which may inhibit their efficacy in combating tumor growth in the brain and other organs. It is therefore important to evaluate the influence of P-gp and BCRP on the pharmacokinetics of anticancer drugs. However, despite clinical trials of CEP-32496 in patients, to our knowledge the potential contribution of P-gp and/or BCRP to resistance against CEP-32496 has not been reported.
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As a molecular imaging modality, positron emission tomography (PET) enables the study of drug pharmacokinetics and pharmacodynamics, and is thus important for both drug discovery and drug development.11 Using radioactive probes in PET aids the study of P-gp/BCRP functions at the blood-brain-barrier (BBB), as well as the evaluation of therapeutic effects of drugs in combating various diseases.12,13 Previously, we have developed the PET probes [11C]gefitinib14,15 and [11C]sorafenib,16 and showed that the pharmacokinetics of both molecular target drugs were significantly affected by both P-gp and BCRP. The aims of this study were to develop [11C]CEP-32496 as a novel PET probe to determine its biodistribution profile in mice and to characterize the influence of P-gp/BCRP-mediated efflux on the uptake of [11C]CEP-32496 in mouse brains. To achieve the radiosynthesis of [11C]CEP-32496, we utilized a reliable method for constructing an unsymmetrical [11C-carbonyl]urea moiety by the reaction of [11C]phosgene ([11C]COCl2)17-19 with two different amines. This approach involves the reaction of an amine hydrochloride with [11C]COCl2, followed by treatment with another free amine to produce the desired unsymmetrical [11C]urea.16,20-22 Here, we applied this technique to synthesize [11C-carbonyl]CEP-32496
(Scheme
1).
CEP-32496
5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine
and
two (1)
precursors: and
3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2), were prepared in house following reported methods,3 with minor modification. The identities of these compounds were confirmed by NMR and high-resolution mass spectra (HRMS).23 After optimizing the reaction conditions, such as reaction temperatures, reaction times, and the amounts of amines 1 and 2 used, we automated the radiosynthesis of [11C]CEP-3249624 with a synthesis system18,19 developed in house. Figure 1A shows a representative HPLC chromatogram for the separation, in which [11C]CEP-32496 was produced as the main radioactive peak in the
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reaction mixture. This protocol yielded 0.76–1.11 GBq of [11C]CEP-32496 at the end of synthesis (EOS), starting with 22–23 GBq of [11C]CO2 (n = 5). The averaged radiochemical yield of [11C]CEP-32496 was 14 ± 3% based on [11C]CO2 (decay-corrected at the end of bombardment (EOB)). The total synthesis time was 35 ± 3 min from EOB.
Figure 1
The identity of [11C]CEP-32496 was confirmed by co-injection with authentic CEP-32496 using analytic HPLC.24 The radiochemical purity of the final [11C]CEP-32496 product in solution was >98% (Figure 1B) and its specific activity was 34–72 GBq/µmol (n = 5) at EOS. No significant UV peaks corresponding to unlabeled 1 and 2 were observed on the HPLC chart. Moreover, the radiochemical purity remained at ~98% after storage at room temperature for 90 min, indicating that [11C]CEP-32496 was radiochemically stable for the duration of at least one PET scan. These analytical results complied with our in-house quality control/assurance specifications.
Table 1
We first studied the biodistribution of [11C]CEP-32496 in the whole bodies of mice (n = 4). Table 1 shows the distribution of radioactivity in mouse tissues following i.v. injection of a saline solution of [11C]CEP-32496 (8.4 MBq/0.1 mL). Radioactivities in tissues are expressed as the percentage of injected dose per gram of wet tissue (% ID/g). High initial radioactivity levels (>10% ID/g) were found in the heart, lung, liver and kidney at 1 min after injection. Radioactivity levels in the blood, heart and lung declined rapidly by 5 min and then decreased slowly over the measurement period of 60 min. The uptake of [11C]CEP-32496 in the liver and pancreas was
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maintained at relatively consistent levels throughout this experiment. Radioactivity gradually accumulated in the small intestine and reached a maximum of 25.4% ID/g at 30 min. These results suggest that the hepatobiliary, urinary excretion and intestinal reuptake pathway dominated the whole-body distribution of [11C]CEP-32496. Compared to peripheral tissues, brain tissues showed low levels of radioactivity uptake (0.21–0.08% ID/g), indicating that [11C]CEP-32496 could not enter the brain easily.
Figure 2
We then carried out a PET study with [11C]CEP-32496 using wild-type and P-gp/BCRP knockout mice under anesthesia with 1–2% isoflurane to compare their relative levels of [11C]CEP-32496 uptake in the brain, which was expressed as the standardized uptake value (SUV).25 Figure 2 shows representative coronal and horizontal PET brain images from wild-type (A, B) and P-gp/BCRP knockout mice (C, D). The radioactivity accumulated in the brains of wild-type mice was markedly lower than that observed with P-gp/BCRP knockout mice. Figure 2E shows time-activity curves in whole brains from both experimental groups. Brains from P-gp/BCRP knockout mice retained high levels of radioactivity, which reached a stable plateau in PET scans between 20 and 60 min after injection of [11C]CEP-32496. Radioactivity levels, measured as areas under the time-activity curves AUC0-90 min (SUV × min) between 0 and 90 min after injection of [11C]CEP-32496, were 49.34 ± 1.5 (n = 3) and 6.1 ± 0.4 (n = 3) for P-gp/BCRP knockout mice and wild-type mice, respectively. The ratio of AUC0-90
min
in the
two-type mice was 8:1. Differences in AUC0-90 min values between the two groups were statistically significant (P < 0.05, Student’s paired t-test).
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Figure 3
To confirm the results of PET study, we harvested blood and dissected brains from P-gp/BCRP knockout and wild-type mice (n = 5 for each group) to determine radioactivity levels (% ID/g) at 60 min after injection of [11C]CEP-32496. As shown in Figure 3, radioactivity uptake in brains from the knockout mice was 24-fold higher than observed with wild-type mice. However, no significant difference was observed in blood uptake levels between the two groups. This study indicates that the uptake of [11C]CEP-32496 in mouse brains may be limited by P-gp and/or BCRP at the BBB. Our results demonstrate for the first time that CEP-32496, like gefitinib and sorafenib, may be a substrate for P-gp and/or BCRP. [11C]CEP-32496 is a promising PET probe that may be used to evaluate and characterize P-gp and/or BCRP functions in vivo. As CEP-32496 is a candidate anticancer drug, PET studies with [11C]CEP-32496 may be useful for BRAF kinase imaging in tumors and for evaluating its bioavailability in combination with other chemotherapeutics against tumors including brain tumors. For example, PET studies may be useful for evaluating the effects of dual P-gp and BCRP inhibitors, such as elacridar,26,27 on the brain permeation with [11C]CEP-32496, in attempt to improve the therapeutic efficacy of CEP-32496 in treating patients with brain tumors. In conclusion, CEP-32496 was successfully labeled with
11
C on its urea group using
[11C]COCl2. [11C-carbonyl]CEP-32496 was produced with sufficiently high specific radioactivity and purity to be acceptable for animal studies. Further studies regarding the effects of P-gp or/and BCRP on the penetration of [11C]CEP-32496 into brain tumors and BRAF kinase imaging in animal models bearing tumors are in progress.
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Acknowledgements We are grateful to Mrs. Y. Kurihara and N. Nobuki (SHI Accelerator Service Co. Ltd.) for providing technical support with radiosynthesis and to Mr. H. Wakizaka (National Institute of Radiological Sciences) for providing assistance with PET scans. We also thank the staff of the National Institute of Radiological Sciences for support in operating the cyclotron operation and the production of radionuclide.
References and Notes 1. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature. 2002, 417, 949. 2. Lee, J. H.; Lee, E. S.; Kim, Y. S. Cancer. 2007, 110, 38. 3. Rowbottom, M. W.; Faraoni, R.; Chao, Q.; Campbell, B. T.; Lai, A. G.; Setti, E.; Ezawa, M.; Sprankle, K. G.; Abraham, S.; Tran, L.; Struss, B.; Gibney, M.; Armstrong, R. C.; Gunawardane, R. N.; Nepomuceno, R. R.; Valenta, I.; Hua, H.; Gardner, M. F.; Cramer, M. D.; Gitnick, D.; Insko, D. E.; Apuy, J. L.; Jones-Bolin, S.; Ghose, A. K.; Herbertz, T.; Ator, M. A.; Dorsey, B. D.; Ruggeri, B.; Williams, M.; Bhagwat, S.; James, J.; Holladay, M. W. J. Med. Chem. 2012, 55, 1082. 4. James, J.; Ruggeri, B.; Armstrong, R. C.; Rowbottom, M. W.; Jones-Bolin, S.; Gunawardane, R.
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N.; Dobrzanski, P.; Gardner, M. F.; Zhao, H.; Cramer, M. D.; Hunter, K.; Nepomuceno, R. R.; Cheng, M.; Gitnick, D.; Yazdanian, M.; Insko, D. E.; Ator, M. A.; Apuy, J. L.; Faraoni, R.; Dorsey, B. D.; Williams, M.; Bhagwat, S. S.; Holladay, M. W. Mol. Cancer Ther. 2012, 11, 930. 5. Carcaboso, A. M.; Elmeliegy, M. A., Shen, J.; Juel, S. J.; Zhang, Z. M.; Calabrese, C.; Tracey, L.; Waters, C. M.; Stewart, C. F. Cancer Res. 2010, 70, 4499. 6. Oostendorp, R. L.; Marchetti, S.; Beijnen, J. H.; Mazzanti, R.; Schellens, J. H. Cancer Chemother. Pharmacol. 2007, 59, 855. 7. Harmsen, S.; Meijerman, I.; Maas-Bakker, R. F.; Beijnen, J. H.; Schellens, J. H. Eur J Pharm Sci. 2013, 48, 644. 8. Gottesman, M. M.; Pastan I. Annu. Rev. Biochem. 1993, 62, 385. 9. Schinkel, A. H.; Wagenaar, E.; van Deemter, L.; Mol, C. A.; Borst, P. J. Clin. Invest. 1995, 96, 1698. 10. Dai, H.; Marbach, P.; Lemaire, M.; Hayes, M.; Elmquist, W. F. J. Pharmcol. Exp. Ther. 2003, 304, 1085. 11. Murphy, P. S.; Bergström, M. Curr. Pharm. Des. 2009, 15, 957 12. Mairinger, S.; Erker, T.; Muller, M.; Langer, O. Curr. Drug Metab. 2011, 12, 774. 13. Yamasaki, T.; Kawamura, K.; Hatori, A.; Yui, J.; Yanamoto, K.; Yoshida, Y.; Ogawa, M.; Nengaki, N.; Wakisaka, H.; Fukumura, T.; Zhang, M.-R. Nucl. Med. Commun. 2010, 31, 985. 14. Kawamura, K.; Yamasaki, T.; Yui, J.; Hatori, A.; Konno, F.; Kumata, K.; Konno, F.; Irie, T.; Fukumura, T.; Suzuki, K.; Kanno, I.; Zhang M.-R. Nucl. Med. Biol. 2009, 36, 239. 15. Zhang, M.-R.; Kumata, K.; Hatori, A.; Takai, N.; Toyohara, J.; Yamasaki, T.; Yanamoto, K.; Yui, J.; Kawamura, K.; Koike, S.; Ando, K.; Suzuki, K. Mol. Imaging. Biol. 2010, 12, 181. 16. Asakawa, C.; Ogawa, M.; Kumata, K.; Fujinaga, M.; Kato, K.; Yamasaki, T.; Yui, J.; Kawamura, K.; Hatori, A.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2011, 21, 2220.
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17. Roeda, D.; Dollé, F. Curr. Top. Med. Chem. 2010, 10, 1680. 18. Ogawa, M.; Takada Y., Suzuki H., Nemoto K., Fukumura T., Nucl. Med. Biol. 2010, 37, 73. 19. Takada, Y.; Ogawa, M.; Suzuki, H.; Nemoto, K.; Fukumura, T. Appl. Radiat. Isot. 2010, 68, 1715. 20. Conway, T.; Diksic, M. J. Nucl. Med. 1988, 29, 1957. 21. Asakawa, C.; Ogawa, M.; Fujinaga, M.; Kumata, K.; Xie, L.; Yamasaki, T.; Yui, J.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2012, 22, 3594. 22. Asakawa, C.; Ogawa, M.; Kumata, K.; Fujinaga, M.; Yamasaki, T.; Xie, L.; Yui, J.; Kawamura, K.; Fukumura, T.; Zhang, M.-R. Bioorg. Med. Chem. Lett. 2011, 21, 7017. 23. Measured properties of the free base of CEP-32496: mp: 203–204 °C; 1H NMR (DMSO-d6, 300 MHz): δ 1.53 (s, 6H, CH3), 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.87 (s, 1H, Ar–H), 6.96–7.00 (m, 1H, Ar–H), 7.25–7.28 (m, 1H, Ar–H), 7.38–7.44 (m, 2H, Ar–H), 7.56–7.58 (m, 2H, Ar–H), 8.56 (s, 1H, Ar–H), 9.01 (s, 1H, Ar–H), 9.74 (s, 1H, Ar–H); HRMS (FAB) calcd for C24H23O5N5F3, 518.1651; found, 518.1606. Measured properties of compound 1: mp: 100–101 °C; 1H NMR (CDCl3, 300 MHz): δ 1.52 (s, 6H, CH3), 4.04 (brs, 2H, NH2), 5.79 (s, 1H, Ar–H); HRMS (FAB) calcd for C7H10ON2F3, 195.0745; found, 195.0758. Measured properties of compound 2: mp: 186–187 °C; 1H NMR (DMSO-d6, 300 MHz): δ 3.97 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 5.28 (brs, 2H, NH2), 6.36–6.43 (m, 2H, Ar–H), 6.46–6.50 (m, 1H, Ar–H), 7.05–7.11 (m, 1H, Ar–H), 7.37 (s, 1H, Ar–H), 7.51 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H); HRMS (FAB) calcd for C16H15O3N3, 297.1113; found, 297.1109. 24. Compound 1•HCl was prepared by mixing 1 (0.61 mg, 3.14 µmol) in THF (500 µL) with 4 M HCl in 1,4-dioxane (4 µL), and [11C]COCl2 gas, which was produced from cyclotron-generated [11C]CO2, was bubbled into the solution at −15 °C for 1 min. After the trapping of [11C]COCl2 was finished, 2 (2.72 mg, 9.15 µmol) and N,N-dimethyl-4-aminopyridine (0.41 mg, 3.36 µmol)
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in THF (200 µL) were added and the reaction mixture was heated to 70 °C for 5 min. Following the removal of THF, the reaction mixture was diluted with HPLC solvent (2 mL) and applied to a reversed-phase HPLC system. HPLC was performed using a JASCO HPLC system (JASCO, Tokyo) and Capcell Pak C18 (Shiseido, Tokyo) columns. A 10 mm × 250 mm column was used for purification, and a 4.6 mm × 250 mm was used for analysis. Ultraviolet detection was performed at 254 nm. A solution of CH3CN/H2O (60/40, v/v) was used as the eluent. Purification was performed using a liquid phase flow rate of 5.0 mL/min for 7.7 min, and analysis was performed using a liquid phase rate of 1.0 mL/min for 7.4 min. The radioactive fraction corresponding to the desired product was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed through a 0.22 µm Millipore filter to give [11C]CEP-32496. 25. PET scans were performed using a small-animal Inveon PET scanner (Siemens, Knoxville, TN), which provides 159 transaxial slices with 0.796 mm (center-to-center) spacing, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial FOV. A bolus of 17–19 MBq of [11C]CEP-23496 (0.44 to 1.08 nmol in 100 µL saline) was injected into the tail vein of mice. Wild-type (male, 18–19 weeks old, 29–31 g, n = 3) and P-gp/BCRP knockout (Abcb1a/1b-/-Abcg2-/-, male, 18–19 weeks old, 35–39 g, n = 3) mice (FVB, Taconic Farm, Hudson, NY) were used. A dynamic emission scan was performed in 3D list-mode for 90 min (1 min × 4 frames, 2 min × 8 frames, 5 min × 14 frames). Region of interest (ROI) analyses and image reconstruction were performed using the ASIPro software (Siemens Medical Solutions). Visual analyses were performed by individuals experienced in PET interpretation, using coronal and horizontal reconstructions. ROIs were manually placed across image planes for time-activity curves. Radioactivity was decay-corrected to the injection time and expressed as a standardized uptake value (SUV) normalized for injected radioactivity and body weight, according to the
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formular: SUV = (radioactivity per cm3 tissue/[injected radioactivity × body weight in grams). The area under the time–activity curve of the ROIs in the brain (AUC0-90 min, SUV × min) was calculated from 0 to 90 min. 26. Lagas, J. S.; Waterschoot, R. A.; Tilburg, V. A.; Hillebrand, M. J.; Lankheet, N.; Rosing, H.; Beijnen, J. H.; Schinkel, A. H. Clin. Cancer Res. 2009, 15, 2344. 27. Kawamura, K.; Yamazaki, T.; Konno, F.; Yui, J.; Hatori, A.; Yanamoto, K.; Wakizaka, H.; Fukumura, T.; Zhang, M.-R. Mole. Imaging Biol. 2011, 13, 152.
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Figure Legends
Scheme 1. Chemical structure of CEP-32496 and radiosynthesis of [11C-carbonyl]CEP-32496: (a) THF, room temperature, 5 min, (b) THF, −15 °C, 1 min; (c) N,N-dimethyl-4-aminopyridine, THF, 70 °C, 5 min, 72% (decay-corrected, analyzed by HPLC for the final reaction mixture).
Figure 1. Chromatograms from the HPLC separation (A) and analysis (B) used in the radiosynthesis of [11C-carbonyl]CEP-32496.
Figure 2. PET imaging of brains in wild-type- and P-gp/BCRP knockout mice. Panels A and B are images of wild-type mouse, and panels C and D are images of P-gp/BCRP knockout mouse. Coronal images are shown in panels A and C, and horizontal images are shown in panels B and D. Panel E shows the time-activity curves in the brains of wild-type- and P-gp/BCRP knockout mice. Data are the means ± SEM (n = 3 for each group).
Figure 3. Radioactivity in the brains and blood of wild-type- and P-gp/BCRP knockout mice at 60 min after injection of [11C]CEP-32496. Data are the means ± SEM (n = 5 for each group).
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Scheme 1. Shimoda et al.
(A) [11C]CEP-32496
(B) [11C]CEP-32496
Figure 1. Shimoda et al.
A
B
E 0.7 0.6 0.5
C
SUV
0.4
D
P-gp/BCRP knockout Wild type
0.3
0
Radioactivity (SUV)
1
0.2 0.1 0.0 0
10
20
30
40 50 min
60
70
80
90
Figure 2. Shimoda et al.
1.6 1.4 P-gp/BCRP knockout Wild type
1.2 % ID/g
1.0 0.8 0.6 0.4 0.2 0.0 Brain
Blood
Figure 3. Shimoda et al.
TABLE 1. Biodistribution in mice Tissue 1 min 5 min 15 min 30 min Blood 3.14 ± 0.25 0.99 ± 0.05 0.95 ± 0.08 0.87 ± 0.03 Heart 11.25 ± 0.46 4.43 ± 0.27 3.56 ± 0.24 3.08 ± 0.08 Lung 14.30 ± 1.16 4.75 ± 0.43 3.76 ± 0.17 3.34 ± 0.09 Liver 17.18 ± 1.82 15.30 ± 0.94 17.40 ± 1.47 18.42 ± 0.44 Pancreas 2.96 ± 0.15 2.77 ± 0.33 3.52 ± 0.14 3.94 ± 0.24 Spleen 3.92 ± 0.28 2.05 ± 0.17 2.05 ± 0.14 2.03 ± 0.05 Kidney 12.33 ± 0.40 8.36 ± 0.80 7.68 ± 0.50 7.44 ± 0.21 Small intestine 5.92 ± 0.51 14.31 ± 1.38 22.18 ± 1.30 25.40 ± 2.10 Large intestine 0.81 ± 0.04 0.76 ± 0.10 1.01 ± 0.08 1.69 ± 0.20 Muscle 2.37 ± 0.15 1.52 ± 0.18 1.60 ± 0.14 1.54 ± 0.05 Brain 0.21 ± 0.02 0.10 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 Data are % injected dose/g wet tissue (% ID/g) (mean ± SEM ; n = 4).
60 min 0.61 ± 0.03 1.98 ± 0.07 2.24 ± 0.09 12.69 ± 0.48 2.66 ± 0.08 1.36 ± 0.05 4.91 ± 0.13 21.23 ± 2.18 1.98 ± 0.06 1.06 ± 0.04 0.08 ± 0.00
Table 1. Shimoda et al.
Graphical abstract
[11C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice
Yoko Shimodaa, Joji Yuia, Masayuki Fujinagaa, Lin Xiea, Katsushi Kumataa, Masanao Ogawaa,b, Tomoteru Yamasakia, Akiko Hatoria, Kazunori Kawamuraa, and Ming-Rong Zhanga*
a)
Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan; b)SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
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