Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain
Introduction
In vivo imaging of GABAA/benzodiazepine receptors in the brain using positron emission tomography (PET) has provided valuable information regarding various neurological conditions and psychiatric disorders. [11C]Flumazenil ([11C]FMZ, ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate, Fig. 1) was the first radioligand developed for in vivo imaging of the benzodiazepine binding site of the GABAA receptor and is currently the most widely used [1], [2]. [11C]FMZ clinical use has been reported in the evaluation of patients with Alzheimer's disease, epilepsy, panic disorders, major depression, cortical brain damage following an acute stroke, anxiety disorders, chronic alcohol dependency, and other brain disorders [3].
There has been extensive evaluation of fluorine-18 labeled agents for GABAA imaging which would circumvent the limitations imposed for [11C]FMZ by eliminating the necessity for an on-site cyclotron, thus providing potential radiopharmaceutical distribution to remote imaging facilities. Flumazenil (Figure 1.1) contains a fluorine substituent, therefore rendering [18F]flumazenil ([18F]FMZ) an alternative to its carbon-11-labeled counterpart. The radiochemical synthesis of [18F]FMZ was first reported using 18F- for-19F exchange [4], [5], and subsequently by nucleophilic aromatic substitution of the meta-nitro precursor with [18F]fluoride ion [6], [7], [8], and via a diaryliodonium precursor [9]. When directly compared to [11C]FMZ in human studies, [18F]FMZ showed equivalent kinetic and metabolic behaviors [10].
In the search for alternative labeled benzodiazepines with improved pharmacokinetics and/or simplified radiochemical syntheses, additional analogs of flumazenil incorporating fluorine-18 have been reported. Replacement of the ethyl ester or the N-methyl group with a 2-[18F]fluoroethyl group yielded the potential radiotracers [18F]FFMZ and [18F]FEFMZ (Fig. 1, compounds 2 and 3) [11], [12]. Neither of these alternative fluorine-18 benzodiazepines however exhibited satisfactory in vivo properties when compared with [18F]FMZ. More recently, Jackson et. al. reported a lengthy series of new FMZ analogs suitable for fluorine-18 labeling [13]. Of particular interest were the analogs where the fluorine substituent was moved from the 8- to the 7-position (Fig. 2). This provided high affinity FMZ analogs (AH114726 (4), Ki = 5.5 nM, and GEH120348 (5), Ki = 0.76 nM, Fig. 2) with simplified radiochemistry. The efficiency of nucleophilic aromatic substitution by [18F]fluoride ion can be improved by placing the leaving group in the position ortho to the carbonyl [14]. In preliminary studies, these new compounds showed good rodent brain uptake, differential binding to GABAA-rich brain regions and a lack of brain metabolites (A. Jackson, GE Healthcare, unpublished results). AH114726 was screened against > 50 different brain receptors, ion channels and transporters using a single point high concentration assay, and 10 μM was found to inhibit 96% of GABAA. No other significant inhibition of other targets was noted. It is also expected that these new compounds will have comparable subunit selectivity comparable to flumazenil [15], [16], but this has yet to be confirmed. The purpose of the present study was to evaluate the in vivo pharmacokinetics of [18F]AH114726 and [18F]GEH120348 in non-human primates, and directly compare such with [11C]FMZ.
Section snippets
Materials
Chemicals and solvents were purchased from Sigma-Aldrich (Milwaukee, WI) or Fisher Scientific (Fair Lawn, NJ) and used without further purification. Unlabeled precursors and reference standards of [19F]GEH120348 and [19F]AH114726 were provided from GE Healthcare (Princeton, NJ). The quality control HPLC column: Luna C18(2) 5 μ 150 × 4.6 mm, and the semi-preparative column: Luna C18 5 μ 250 × 10 mm were purchased from Phenomenex (Torrance, CA). For preparation of [18F]GEH120348 and [18F]AH114726,
Radiochemistry
The syntheses of [18F]GEH120348 and [18F]AH114726 were achieved by direct nucleophilic aromatic fluorination by substituting the nitro group on precursors 6 or 7 with [18F]fluoride (Scheme 1). [18F]GEH120348 and [18F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol).
Primate microPET imaging
Representative coronal images of control animals (Fig. 3, top row), and after displacement with unlabeled
Discussion
The 7-fluoro imidazobenzodiazepines evaluated in this study were developed as alternative structures for synthesis of fluorine-18 labeled radioligands for in vivo imaging of the GABAA/benzodiazepine receptor system. The simplified radiochemical syntheses worked as expected, proving reliable and giving high yields of [18F]AH114726 and [18F]GEH120348 (13–24% at end of synthesis based upon starting fluoride). Amounts prepared were hundreds of millicuries, confirming the suitability of the
Conclusion
The 7-[18F]fluoro imidazobenzodiazepine analogs of FMZ are suitable alternatives to the 8-[18F]fluoro imidazobenzodiazepine derivatives (such as [18F]FMZ, [18F]FFMZ, and [18F]FEFMZ). The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [11C]flumazenil, support [18F]AH114726 as a suitable tracer for imaging the GABAA/benzodiazepine receptor.
Acknowledgments
Research reported in this publication was supported in part by the National Institute of Biomedical Imaging and Bioengineering, part of the National Institutes of Health, under Award Number T32-EB005172. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional financial support from GE Healthcare is also gratefully acknowledged. Finally, the authors thank Ian Wilson and Matthew S. Morrison for
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