MATERIALS AND METHODS
Sodium hydroxide (NaOH), chloramine T, sodium disulfite (Na2S2O5), acetonitrile, and tetrahydrofuran (THF) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). 5-Chloro-7-iodo-8-quinolinol (clioquinol), and 5-chloro-8-quinolinol (cloxyquin) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, U.S.A.). Sodium dihydrogen phosphate (NaH2PO4) and zinc acetate dihydrate ((CH3COO)2Zn·2H2O) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Zinc chloride (ZnCl2) was purchased from Tokyo Kasei Inc. (Tokyo, Japan). Iodine-131 radionuclide (131I) was obtained from PerkinElmer, Inc. (MA, U.S.A.).
Male KK-Ay mice were purchased from CLEA Japan, Inc. All animals were housed under a 12-hour light/dark cycle in a temperature-controlled animal room and allowed free access to food and tap water. All animal experiments were approved by the Ethics Committee on Animal Care and Use of RIKEN and Okayama University, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Synthesis of [Zn(Cq)2]
(CH3COO)2Zn·2H2O (329 mg, 1.5 mmol) in water (4.0 mL) was added dropwise to clioquinol (5-chloro-7-iodo-8-quinolinol) (Cq) (611 mg, 2.0 mmol) in THF (10.0 mL), and the reaction mixture was stirred at room temperature for 90 min. The yellow precipitate was filtered, rinsed with acetone, and evaporated in a vacuum to obtain the pure product as a yellow solid. Yield: 523 mg (77.6%); 1H-NMR (dimethyl sulfoxide (DMSO)-d6), δ: 7.69 (1H, dd, J=4.2, 8.4 Hz), 7.90 (1H, s), 8.45 (1H, dd, J=4.2, 8.4 Hz), 8.47 (1H, dd, J=4.2, 4.2 Hz); electron ionization (EI)-MS m/z: 674 (M+) Anal. Calcd for C18H8Cl2I2N2O2Zn: C, 32.05; H, 1.19; N, 4.15. Found: C, 32.31; H, 1.05; N, 4.11.
Synthesis of 65Zn-, and 131I-Labeled [Zn(Cq)2]
No-carrier-added Na131I (14 MBq) in 0.1 M NaOH (20 µL) was added to cloxyquin (20 µg, 0.11 µmol) in ethanol (25 µL). The solution was buffered to pH 5 by adding 20 µL 0.1 M NaH2PO4. A solution of chloramine-T (20 µg, 0.071 µmol) in water (8 µL) was then added and the reaction mixture allowed to stand at room temperature for 2 min. After quenching the reaction with Na2S2O5 (100 µg, 0.66 µmol) in water (20 µL), the mixture was purified by reverse phase (RP)-HPLC using a COSMOSIL 5C18-AR-ΙΙ, 5 µm, 4.6×150 mm column (Nacalai Tesque Inc.). A mobile phase of acetonitrile/0.1% TFA in water (80/20%, v/v) was used and the flow rate was 0.10 mL/min. The radioactivity peak corresponding to [131I]Cq was collected between 4.2 and 5.2 min in a volume of 0.10 mL. The [131I]Cq solution was evaporated and dissolved in THF. The resultant solution was mixed with non-radiolabeled Cq in THF.
The 65Zn nuclide was produced by a previously described method.14) The purified 65Zn in HNO3 was dried using a heater, and the residue was then dissolved in acetic acid. The solution was dried again and dissolved in water. The resulting solution was mixed with (CH3COO)2Zn·2H2O aqua. (CH3COO)2Zn·2H2O (3.58 mg, 16.3 mmol) containing 65Zn (8.28 MBq) in water (110 µL) was added dropwise to carrier-added [131I]Cq (9.97 mg, 16.3 mmol, 3.43 MBq) in THF (250 µL), and the reaction mixture was stirred at room temperature for 90 min. The yellow precipitate was filtered, rinsed with acetone, and dried up in a vacuum to yield 10.2 mg (92.7%) of [Zn(Cq)2] containing 7.04 MBq of 65Zn (85.0%) and 2.82 MBq of 131I (82.4%). Chemical structure of 65Zn- and 131I-labeled [Zn(Cq)2] is shown in Fig. 1. Before the administration, non-radiolabeled [Zn(Cq)2] was added to 65Zn-, 131I labeled [Zn(Cq)2], and dissolved in PEG400. ZnCl2 was dissolved in PEG400 containing 5% water.
|Fig. 1. Chemical Structure of 65Zn- and 131I-Labeled [Zn(Cq)2]|
Nine-week-old male KK-Ay mice were orally administered a single dose of 65Zn-, 131I labeled [Zn(Cq)2] or [65Zn]ZnCl2 at a dose of 10 mg Zn/kg of body weight (65Zn: 150 kBq, 131I: 50 kBq per head). Two or six hours after administration, the mice were sacrificed under isoflurane anesthesia. Blood was collected, and the organs (heart, stomach, pancreas, liver, small intestine, kidney, thigh muscle, femur bone, and adipose tissue) were removed. Their radioactivities due to 65Zn and 131I were measured using a calibrated Ge detector or gamma counter. Results were expressed as percent injected dose per gram of tissues (%ID/g).
Nine-week-old male KK-Ay mouse was orally administered a single dose of 65Zn-, 131I labeled [Zn(Cq)2] at 10 mg Zn/kg of body weight (65Zn: 1.5 MBq, 131I: 0.5 MBq per head). Fifteen minutes after administration, the GREI experiments were carried out under isoflurane anesthesia for 30 min, and for 60 min at 2, 4, 6, and 24 h after administration. The acquired data were recorded in list mode with real-, and live-time information. The distribution images were reconstructed from the acquired data by the adoption of the image-reconstruction methods as previously described.18) The mouse was sacrificed under isoflurane anesthesia right after the GREI experiments at 24–25 h. Blood was collected, and the organs described above in addition to the large intestine were removed. The radioactivity in each tissue was determined using a Ge detector. The radioactivities in the stomach, small intestine, and large intestine were measured with or without their contents. Results were expressed as percent injected dose (%ID).
To evaluate the biodistribution of 65Zn and 131I and the speciation of [Zn(Cq)2] noninvasively, regions of interest (ROIs) were drawn around the radioactivity accumulated areas. The radioactivity in each ROI was calculated by integrating the pixel values inside them. In the ROI analysis, the whole body radioactivity at 0–0.5 h after an administration was taken as the administered radioactivity, because the images were acquired before excretion. The ratio of radioactivity in each ROI to the administered radioactivity was calculated for 65Zn and 131I, respectively. The I/Zn ratio that corresponded to the molar ratio of administered I to Zn was calculated by following equation:
are the image intensity of 131
I in each ROI and in the whole body at 0–0.5 h after an administration, respectively, and ZnROI
are the image intensity of 65
Zn in each ROI and in the whole body at 0–0.5 h after an administration, respectively. The minimum dissociation rate (MDR) of [Zn(Cq)2
] was calculated from the following equation;