CHAPTER 1
SYNTHESIS OF (-)-[18F]FLUBATINE ([18F]FLBT)

MEGAN N. STEWART, BRIAN G. HOCKLEY, AND PETER J. H. SCOTT

Department of Radiology, University of Michigan School of Medicine, Ann Arbor, Michigan, USA

1 INTRODUCTION

Cognitive or depressive disorders and neurodegenerative diseases such as Alzheimer's disease (AD), dementia, and Parkinson's disease (PD) may be related to dysfunctional signaling through a4ß2-nicotinic acetylcholine receptors (a4ß2-nAChRs) [1, 2]. Alterations in the cholinergic system are also implicated in the progression of cognitive decline in the aforementioned neurodegenerative diseases, particularly AD [2-4]. The development of (-)-[18F]flubatine as a high affinity and selective PET radiotracer with improved kinetics over the earlier developed ligands allows for noninvasive quantification of nAChRs [2, 5].

The first reported radiosynthesis of (-)-[18F]flubatine, a derivative of epibatidine, utilized a norchloro-bromo-homoepibatidine (NCBrHEB) precursor that underwent a nucleophilic substitution with the bromine leaving group, then the enantiomers separated, and the product purified appropriately via HPLC [4-7]. However, due to low radiochemical yields, other candidate precursors were explored for radiolabeling and the trimethylammonium iodide-Boc-protected compound ((5-((1R,5S,6S)-8-tert-butoxycarbonyl)-8-azabicyclo[3.2.1]-octan-6-yl)-N,N,N-trimethylpyridin-2-aminium iodide, Boc-trimethylammonium homoepibatidine, 1) was shown to give the best yields of approximately 60% and further adapted for fully automated synthesis [2, 8]. This precursor has since become commercially available, making [18F]flubatine more accessible for clinicians and has been validated for clinical use in nonhuman primates [4].

2 SYNTHESIS PROCEDURES

CAUTION: All radiochemical syntheses must be carried out using appropriate equipment in a facility authorized for the use of radioactive materials. Personal protective equipment must be worn and all local radiation safety laws followed.

2.1 Production of [18F]Fluoride

[18O]H2O (1.5?ml) [9] was loaded into the [18F]fluoride target [10] of a General Electric Medical Systems (GEMS) PETtrace cyclotron [11]. The target was bombarded (60?µA beam for 30?min) to generate approximately 1.5?Ci (55.5?GBq) of [18F]fluoride by the 18O(p,n)18F nuclear reaction.

2.2 Azeotropic Drying of [18F]Fluoride

The [18F]fluoride was delivered to a GEMS TRACERlab FXFN synthesis module [11] as a solution in [18O]H2O (1.5?ml). This solution was passed through a Sep-Pak® QMA-Light cartridge [12] to trap the [18F]fluoride and recycle the [18O]H2O. The [18F]fluoride was then eluted into the TRACERlab FXFN glassy carbon reaction vessel using a solution of aqueous potassium carbonate (3.5?mg in 0.5?ml H2O) [13]. A solution of Kryptofix 222 (5?mg in 1?ml MeCN) [14] was added and the reaction mixture was azeotropically dried, initially at 80°C under vacuum for 4?min and subsequently at 60°C with both vacuum and argon flow for an additional 4?min.

2.3 Synthesis of (-)-[18F]FLBT

A solution of (-)-FLBT precursor [15] (1, 0.5-1.0?mg) in anhydrous dimethyl sulfoxide (DMSO) [16] (0.6?ml) was added to the dried [18F]fluoride, and the reaction was heated to 120°C with stirring for 10?min (Fig. 1). After this time, the reaction was cooled to 40°C, and 1.0?M aqueous hydrochloric acid (1?ml) was added. The reaction was stirred for 5?min at 80°C to hydrolyze the Boc protecting groups. The reaction mixture was neutralized with 0.5?M aqueous sodium hydroxide (2?ml) [17].

FIGURE 1 Synthesis of (-)-[18F]FLBT.

2.4 Purification and Formulation of (-)-[18F]FLBT

After hydrolysis, the crude reaction mixture was purified by semipreparative HPLC (Luna 10u C18(2) 250?×?10?mm column [18] , flow rate?=?4?ml/min), and a representative HPLC trace is shown in Fig. 2.

FIGURE 2 Semipreparative UV and radioactive HPLC traces for [18F]FLBT.

The fraction corresponding to (-)-[18?F]FLBT (typically eluting between 20 and 25?min) was collected for 1?min into a vial precharged with 0.9% sodium chloride, USP [19] (6?ml). The final formulation (10?ml) was then passed through a sterile filter [20] into a sterile vial [21] to provide (-)-[18F]FLBT (typically 50-115?mCi (1.85-4.3?GBq)) in an isotonic solution released for quality control. After synthesis was complete, the semipreparative HPLC column was flushed with 70% ethanol.

3 QUALITY CONTROL PROCEDURES

CAUTION: All radiochemicals produced for clinical use must have local regulatory approval (e.g., FDA, EMEA, MHRA, PFSB, etc.) prior to human use. Quality control procedures must be carried out by trained personnel, and each dose must meet all established QC criteria before release to the clinic.

Quality control (QC) procedures for (-)-[18F]FLBT, based upon the current requirements for radiopharmaceuticals laid out in the US Pharmacopeia (USP) [22] , are summarized in the following text. Complete QC data for three repeat batches of (-)-[18F]FLBT produced using the method disclosed herein are summarized in Table 1. Each of the three doses met all of the established QC criteria

TABLE 1 QC Data for three Repeat Runs of (-)-[18F]FLBT

3.1 Visual Inspection

The (-)-[18F]FLBT dose is examined behind a PET L-block and must be clear, colorless, and free of particulate matter.

3.2 Radiochemical Identity

HPLC analysis of radiochemical identity was conducted using a Shimadzu LC-2010AHT Liquid Chromatograph [23] fitted with UV detectors and Bioscan ?-detectors [24] (column, Phenomenex Synergi Polar RP 150?×?4.6?mm [25]; mobile phase, 50% acetonitrile 50% water +0.1% acetic acid [26]; flow rate, 1?ml/min, ??=?254?nm). The retention time of [18F]FLBT is compared to that of the [19F]FLBT reference standard [15] and must be ±10% (relative retention time (RRT) must be 0.9-1.1).

3.3 Radiochemical Purity

HPLC analysis of radiochemical purity was conducted using a Shimadzu LC-2010AHT Liquid Chromatograph [23] fitted with a UV detector and a Bioscan ?-detector [24] (column, Phenomenex Synergi Polar RP 150?×?4.6?mm [25]; mobile phase, 50%...