Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

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Abstract

Introduction

Two 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABAA/benzodiazepine receptor by comparing them to [11C]flumazenil in rhesus monkey.

Methods

Radiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [18F]AH114726 and [18F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.

Results

[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). The in vivo pharmacokinetics of [18F]AH114726 and [18F]GEH120348 were determined in a non-human primate and directly compared with [11C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [11C]flumazenil and the known concentrations of GABAA/benzodiazepine receptors in the monkey brain. [18F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [11C]flumazenil. In contrast, [18F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.

Conclusion

The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [11C]flumazenil, support that [18F]AH114726 is a suitable option for imaging the GABAA receptor.

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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