ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 8, No. 1, 17-22 1994 

11C-Labeled 2'-iododiazepam for PET studies of benzodiazepine receptors: Synthesis and comparison of biodistribution with its radioiodinated compound 

Yasuhiko IIDA,* Hideo SAJI,* Yasuhiro MAGATA,** Junji KONISHI,** Iwao NAKATSUKA,*** Akira YOSHITAKE*** and Akira YOKOYAMA* 

*Faculty of Pharmaceutical Sciences, **School of Medicine, Kyoto University ***Environmental Health Sciences Laboratory, Sumitomo Chemical Co. Ltd. 

For PET studies of benzodiazepine receptors, N-11C-methyl-2'-iododiazepam (2'-IDZ) was synthesized by N-methylation of its desmethyl derivative with 11C-methyl iodide, and was subsequently purified by HPLC. The labeling and purification procedures were completed within 45 min after 11C-methyl iodide trapping, and the radiochemical yield (corrected for decay) was approximately 40% based on the initial trapped radioactivity of 11C-methyl iodide. Biodistribution studies in mice demonstrated that 11C-2'-IDZ was rapidly and noticeably accumulated in the brain, and subsequently decreased with time. Accumulation was greater in the cortex than in other brain regions. When compared with 125I-2'-IDZ, the distribution was almost the same until 5 min after injection, but levels were low after 20 min. Metabolic studies indicated that the difference between these two compounds in the time course of brain radioactivity distribution may be due to N-demethylation in vivo. 

Key words: diazepam derivative, 11C, 125I, benzodiazepine receptor, PET 

INTRODUCTION 
ALTERATIONS in the biochemical integrity of the benzodiazepine receptor system have been related to the development and evolution of various neurological and psychiatric disorders, including epilepsy, 1,2 Huntington's disease, 3,4 and Alzheimer's disease. 5 The in vivo imaging of benzodiazepine receptors by positron emission tomography (PET) and single photon emission tomography (SPECT) has therefore been a growing field of interest. 6-9 For such studies, a ligand capable of being labeled with both positron- and single photon-emitting radionuclides would be useful, since it would then become possible to investigate benzodiazepine receptors by PET and SPECT with a single radioligand and to compare both imaging methods directly 10-12. 
The basic requirements of a radioligand for in vivo studies of benzodiazepine receptors include rapid and 

Received May 26, 1993, revision accepted September 3, 1993. 
For reprint contact: Hideo Saji, Ph.D., Department of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01 , JAPAN. 

quantitatively significant brain uptake following peripheral administration and a high affinity for the target receptors. 13-15 1,4-Benzodiazepine compounds such as diazepam show signs of high extraction by the brain. 6-9,16 In addition, structure-activity studies have indicated that the introduction of electron-withdrawing groups such as halogens at position 2' of the 5-phenyl-1,4-benzodiazepine molecule increases its receptor affinity. 17-20
Accordingly, we recently developed a diazepam derivative, 2'-iododiazepam (2'-IDZ), which has both a methyl group at the N-1 position as a site for introducing 11C and an iodine atom at the 2' position as the site for introducing 123I (Fig. 1 ). 21,22 This compound was found to have a high affinity for cerebral benzodiaze pine receptors, approximately 9 times greater than that of diazepam (Kd = 0.66 nM). 21,22 In addition, radioiodinated 2'-IDZ was synthesized and was found to bind to benzodiazepine receptors in vivo. 22 In the present study, 11C-labeled 2'-IDZ was synthesized, and its in vivo behavior was compared with that of its 125I-labeled analog. In addition, its usefulness for imaging cerebral benzodiazepine receptors was assessed.

MATERIALS AND METHODS 
125I-2'-IDZ was prepared by a bromine-iodine exchange reaction with sodium 125I-iodide (Amersham International Plc.), as described previously. 21,22 All other chemicals used were of reagent grade. Male ddY mice were supplied by Japan SLC Co. Ltd. 

Preparation of 11C-2'-IDZ 
11C-Methyl iodide was prepared according to the method described previously. 23 11C-Carbon dioxide was produced via proton bombardment of nitrogen gas and the 14N(p,alpha)11C reaction in an ultracompact cyclotron (Sumitomo, Model 325), and was trapped in a solution of lithium aluminum hydride in tetrahydrofuran (THF). After evaporation of the THF, 54% hydroiodic acid was added, and the 11C-methyl iodide thus produced was trapped in an acetone solution (350 micro l) containing 0.4 mg of 2'-iodonordiazepam (IND) and 5 micro l of 10 N sodium hydroxide. Then the reaction vial was sealed and heated at 90deg.C for 8 min. After cooling and neutralization of the solution with 1 N hydrochloric acid, the resulting mixture was purified by high-performance liquid chromatography (HPLC) on a 7.5 x 300 mm Lichrosorb RP-18 column eluted with ethanol : water (9 : 11) at 1.5 ml/min (Rt = 25 min for 2'-IND; Rt = 30 min for 2'-IDZ). The fraction corresponding to 2'-IDZ was collected and evaporated, after which the residue was dissolved in ethanolic saline (< 0.5% ethanol) and filtered through a 0.22 micro m cellulose acetate filter (Milex, Millipore Corp.). 
The radiochemical purity of the product was determined by thin-layer chromatography (TLC) and analytical HPLC. TLC was performed on a silica gel plate with a chloroform : acetone (4 : 1) solvent (Rf = 0.62 - 0.68). HPLC was performed on a 7.5 x 300 mm Lichrosorb RP-18 column eluted with methanol : water : 4 mM phosphate buffer (pH 7.4) (11 : 5 : 4) at a flow rate of 1.5 ml/ min (Rt = 62 min). The specific activity was estimated from the U.V. absorbance at 315 nm. 
2'-IND was synthesized from 2-iodobenzonitrile by the addition of 4-chloroaniline, followed by coupling with bromoacetyl chloride, amination, and cyclization. The structure of the compound was confirmed by infrared analysis (IR), proton nuclear magnetic resonance (NMR) spectrometry, and mass spectrometry. Details of the synthesis and characterization of this compound will be published elsewhere. 

Biodistribution study in mice 
Male ddY mice weighing about 30g were injected via the tail vein with 11C-2'-IDZ ( 1.11 MBq, 3.1 micro g/kg) or 125I-2'-IDZ ( 18.5 kBq, 0.03 micro g/kg) in 0.1 ml of ethanolic saline. At designated times afterwards, the mice were killed by decapitation and their organs were removed. For in vivo brain distribution studies, the brains were dissected on an ice-cold plate according to the method of Glowinski and Iversen. 24 All samples were weighed, and the radioactivity was counted in an NaI well scintillation counter. Results are presented as the % injected dose/g tissue weight. 

Metabolic study 
Mice weighing about 30 g were injected intravenously with 18.5 MBq of 11C-2'-IDZ or 55.5 kBq of 125I-2'-IDZ and then decapitated at 5 or 20 min after injection. The brains were removed immediately and homogenized in 1 ml of methanol. After centrifugation, the precipitate was washed twice with 1 ml of methanol and the washings were combined with the supernatant. For both compounds, approximately 95% of the radioactivity in the homogenate was extractable by our organic solvent technique. The combined methanol extracts were evaporated, and the resultant residue was redissolved in a small volume of methanol for analysis by HPLC. HPLC was performed as described above on a Lichrosorb RP-18 column. 

RESULTS AND DISCUSSION 
11C-2'-IDZ was prepared by N-methylation of 2'-iodo-nordiazepam (2'-IND) with 11C-methyl iodide, and was well separated from the starting material (2'-IND) and some radioactive by-products by reverse-phase HPLC. Radioactivity due to compounds other than 11C-2'-IDZ (including 11CH3I) was detected in the early fractions. The labeling and purification procedures were completed within 45 min after the finish of 11C-methyl iodide trapping and the radiochemical yield (corrected for decay) was approximately 40% based on the initial radioactivity of 11C-methyl iodide. Radiochemical purity of the product was greater than 98% as determined by HPLC and TLC. The specific activity was 3.7-4.8 TBq/mmol at the end of synthesis when 2.6 MBq of 11C-methyl iodide was utilized in the procedure. In the biodistribution study, 11C-2'-IDZ was found to rapidly enter the brain, and high uptake was observed during the early phase, after which the brain levels declined with time (Table 1 ). The radioactivity was cleared rapidly from the blood, and the highest brain-to-blood ratio of 3.3 was obtained at 5 min after injection. At the initial sampling point of 1 min, there was a higher uptake in the heart and lungs than in the brain, but the radioactivity in these organs fell below the brain level at 5 min after injection. The kidneys also showed a high initial uptake, but the clearance of radioactivity was slower than in the heart and lungs. If the distribution of a compound is only dependent on blood perfusion, the organs with a high uptake show more rapid washout. 25 Thus, the results obtained in this study suggest that the distribution of 11C-2'-IDZ was not only dependent on perfusion. In addition, the liver showed a rapid increase in radioactivity, which reached a maximum at 5 min after injection, and then gradually decreased with time. 
The biodistribution of 11C-2'-IDZ was almost the same as that of 125I-2'-IDZ within 5 min after injection. However, the levels of 11C-2'-IDZ in all organs tested were lower than those of 125I-2'-IDZ in the late phase (Fig. 2). 
In the liver, diazepam and its analogs are known to be rapidly metabolized to 3-hydroxylated and N-demethylated compounds, which are still pharmacologically active and can redistribute in other organs. 16,19,20,26-29 In contrast, it has been reported that diazepam is not metabolized in the brain. 29 If 2'-IDZ was metabolized in a manner similar to diazepam, it would be transformed to 3-hydroxylated and N-demethylated analogs in the liver (Fig. 3). 3-Hydroxylation is unlikely to cause the loss of radioactivity from either 11C-2'-IDZ or 125I-2'-IDZ. On the other hand, it can be predicted that, after N-demethylation, 11C-N-methylated 2'-IDZ would lose its radioactivity by excretion through the lungs as 11C02, while 125I-2'-IDZ would be transformed in the liver to an N-demethylated metabolite that traps its radioactivity and is distributed to some other organs including the brain (Fig. 3). 25,29,30 Thus, the difference in the biodistribution of these two compounds may be due to their metabolism by N-demethylatlon. In fact, radio-HPLC analysis (corrected for decay) of brain homogenates obtained at 20 min after radioligand injection showed that, apart from the hydrophilic metabolite (Rt = 8 min), the number of major metabolites was one (Rt = 42 min) and two (Rt = 42 min and 44 min) for 11C-2'-IDZ and 125I-2'-IDZ, respectively (Fig. 4). The metabolites eluted at 42 min and 44 min would correspond to the 3-hydroxy and N-demethyl analogs, respectively, as judged from their retention times on HPLC. On the other hand, both compounds showed the same HPLC profiles at 5 min after injection, at which time most of the radioactivity (> 94%) in the brain homogenates remained as 2'-IDZ. These results indicate that this compound enters the brain in its unchanged form soon after administration. 
The regional distribution of radioactivity in the mouse brain is shown in Table 2. The cortex tended to show a higher uptake of radioactivity than the other brain regions until 5 min after injection, although the regional variation in distribution became small after 20 min. This differential regional distribution during the early period paralleled the distribution of benzodiazepine receptors shown by previous in vitro and PET studies, 6,8,9.31,32 suggesting that 11C-2'-IDZ bound to benzodiazepine receptors in the brain.
In conclusion, 11C-2'-IDZ showed prominent uptake by the brain regions containing benzodiazepine receptors soon after injection, and therefore appears to be a potentially useful radiopharmaceutical for PET studies of cerebral benzodiazepine receptors. In addition, our results suggested that 2'-IDZ had the potential for use in direct comparative PET and SPECT imaging for the localization and quantification of benzodiazepine receptors with a single tracer with different radioactive moieties (i.e., 11C-2'-IDZ and 123I-2'-IDZ). 

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