Radiodefluorination of 3-Fluoro-5-(2-(2-[F](fluoromethyl)- thiazol-4-yl)ethynyl)benzonitrile ([F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2[F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [F]fluoride ion from [F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M H] of S-glutathione (SP203Glu) and N-acetyl-S-Lcysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M H] of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand. Metabotropic subtype-5 receptors (mGluR5) have a discrete distribution in brain and are a potential target for treating several neuropsychiatric disorders, including schizophrenia (Pietraszek et al., 2007), Alzheimer’s disease (Tsai et al., 2005), anxiety (Brodkin et al., 2002), depression (Li et al., 2006), drug addiction (Kenny and Markou, 2004), and fragile X syndrome (Dölen et al., 2007). Potent noncompetitive mGluR5 antagonists, such as 6-methyl-2-(phenylethynyl)pyridine (Gasparini et al., 1999) and 3-[(2-methyl-1,3thiazol-4-yl)ethynyl]pyridine (Cosford et al., 2003), have been developed. Nevertheless, a useful drug is yet to emerge from these antagonists for treating any of the implicated neuropsychiatric disorders. Selective imaging of mGluR5 in living human brain with a suitable radioligand and a technique such as positron emission tomography (PET) is needed to This work was supported by the Intramural Research Program of National Institutes of Health (National Institute of Mental Health project Z01-MH002795-04). A portion of this work was presented at the 55th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, 2007 Jun 3–7, Indianapolis, IN. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.143347. ABBREVIATIONS: mGluR5, metabotropic glutamate subtype-5 receptor(s); PET, positron emission tomography; SP203, 3-fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile; [F]FCWAY, F-trans-4-fluoro-N-(2-[4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-N-(2-pyridyl)cyclohexanecarboxamide; [F]SP203, 3-fluoro-5-(2-(2-[F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; MS/MS, mass spectrometry/mass spectrometry; amu, atomic mass unit(s); MS, multi-stage mass spectrometry; SUV, standardized uptake value; CID, collision-induced dissociation. 0022-3565/08/3273-727–735 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 327, No. 3 U.S. Government work not protected by U.S. copyright 143347/3411023 JPET 327:727–735, 2008 Printed in U.S.A. 727 http://jpet.aspetjournals.org/content/suppl/2009/01/22/jpet.108.143347.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M ay 6, 2017 jpet.asjournals.org D ow nladed from better elucidate the role of mGluR5 in health and neuropsychiatric disorders and thus to facilitate drug discovery. Successful PET imaging and quantification of brain mGluR5 depends on the properties of the radioligand in vivo, especially receptor affinity, metabolism, and blood-brain barrier permeability (Pike, 1993; Laruelle et al., 2003). We have discovered a high-affinity ligand, SP203 (Fig. 1) and carried out labeling with [F]fluoride ion in one step from its bromomethyl analog to generate [F]SP203 (Lazarova et al., 2007; Sharma and Lindsley, 2007; Siméon et al., 2007). PET evaluation of [F]SP203 in monkey demonstrated that a high proportion of radioactivity in brain was bound to mGluR5. However, radioactivity also accumulated in bone, showing metabolism and release of [F]fluoride ion from [F]SP203. The accumulation of radioactivity in bone, especially skull, would be troublesome for the quantification of mGluR5 in monkey brain, because of errors derived from the significant partial volume effects that are associated with the limited multimillimeter spatial resolution of PET. Here, we found that the radiodefluorination of [F]SP203 could not be inhibited with disulfiram, a potent inhibitor of CYP2EI, as we had shown previously to be effective for preventing the defluorination of another PET radioligand, [18F]FCWAY (Ryu et al., 2007). The main purpose of this study was to examine the metabolism of [F]SP203/SP203 in rat so as to identify a biochemical basis for radiodefluorination of the radioligand. We conclude that nucleophilic substitution with glutathione in [F]SP203 at its [F]fluoromethyl group is responsible for releasing [F]fluoride ion. Materials and Methods