Radiosynthesis and Evaluation of [ 11 C]3-Hydroxycyclopent-1- enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ‑Hydroxybutyric Acid Binding Sites Claus H. Jensen, § Hanne D. Hansen, † Tina Bay, § Stine B. Vogensen, § Szabolcs Lehel, ‡ Louise Thiesen, § Christoffer Bundgaard, ∥ Rasmus P. Clausen, § Gitte M. Knudsen, † Matthias M. Herth, §,†,‡ Petrine Wellendorph, § and Bente Frølund* ,§ § Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark † Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging, Rigshospitalet and University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark ‡ PET and Cyclotron Unit, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark ∥ Discovery DMPK, H. Lundbeck A/S, Ottiliavej, 2500 Valby, Denmark * S Supporting Information ABSTRACT: γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specificaffinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specificaffinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the 11 C-labeling and subsequent evaluation of [ 11 C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [ 3 H]HOCPCA. In vivo evaluation of [ 11 C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [ 11 C]HOCPCA. KEYWORDS: GHB, HOCPCA, GHB binding site distribution, PET, in vitro autoradiography γ-Hydroxybutyric acid (GHB, Figure 1) is an endogenous neuroactive substance that is present in micromolar concen- trations in the mammalian brain. 1 The primary source of GHB is believed to be metabolic derivation from the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) (Figure 1). 2 Additionally, GHB is a recreational drug (Fantasy or liquid ecstacy), but also a clinically prescribed drug for treatment of alcohol dependence (Alcover) 3 and narcolepsy (Xyrem, sodium oxybate). 4 In the central nervous system (CNS), GHB binds to specific high-affinity binding sites that are abundantly expressed and conserved through evolution. 5 The functional role of these has been a matter of thorough investigation for several years. 5 Although many of the observed in vivo pharmacological effects of GHB are mediated by the metabotropic GABA B receptors, the high-affinity GHB binding sites are preserved in brains of GABA B(1) knockout mice, providing evidence that the high- affinity GHB binding sites are molecularly distinct from the GABA B receptor complex. 6 Efforts to uncover their molecular identity are of high interest and have so far been largely driven by the advent of nanomolar affinity and highly selective compounds and radioligands. 7−10 Recently, the α 4 β 1−3 δ ionotropic GABA A receptors have been identified as potential high-affinity GHB targets in vitro, 11 which however remains to be validated by in vivo studies. 12 α 4 knockout mice have ∼40% high-affinity GHB binding sites of wild-type mice, which leave the identity of the remaining 60% of the high-affinity binding sites elusive. 5,11 So far, all these binding studies have exclusively been performed in vitro. Positron emission tomography (PET) is a noninvasive technology and has been highly useful to quantify neuro- receptor binding in vivo. 13 In order to attain a more complete understanding of the identity and physiological relevance of high-affinity GHB binding sites in the CNS, the availability of a suitable PET radiotracer for visualization of these sites would be of significant value. We have previously described the conformationally restricted GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1, Figure 1) as a highly selective ligand for the high-affinity GHB binding site (K i 16 μM) 10 (27 times higher affinity than GHB itself), and, more importantly, with no Received: September 30, 2016 Accepted: November 14, 2016 Published: November 14, 2016 Letter pubs.acs.org/chemneuro © 2016 American Chemical Society 22 DOI: 10.1021/acschemneuro.6b00335 ACS Chem. Neurosci. 2017, 8, 22−27