Research article
Radiosynthesis and biodistribution of a histamine H3 receptor antagonist 4-[3-(4-piperidin-1-yl-but-1-ynyl)-[11C]benzyl]-morpholine: evaluation of a potential PET ligand

https://doi.org/10.1016/j.nucmedbio.2006.05.008Get rights and content

Abstract

The potent histamine H3 receptor antagonist JNJ-10181457 (1) was successfully labeled with 11C in a novel one-pot reaction sequence, with high chemical yield (decay-corrected yield, 28±8%) and high specific radioactivity (56±26 GBq/μmol). The binding of [11C]1 to H3 receptors was studied in vitro in rat brain and in vivo in rats and mice. The in vitro binding of [11C]1 in rat coronal brain slices showed high binding in the striatum, and this binding was blocked by histamine and by two known H3 antagonists, JNJ-5207852 (2) and unlabeled Compound (1), in a concentration-dependent manner. The biodistribution of [11C]1 in rats was measured at 5, 10, 30 and 60 min. The uptake of [11C]1 in regions rich in H3 receptors was highest at 30 min, giving 0.98%, 1.41%, 1.28% and 1.72% dose/g for the olfactory bulb, hippocampus, striatum and cerebral cortex, respectively. However, the binding of [11C]1 in the rat brain could not be blocked by pretreatment with either Compound (2) (30 min or 24 h pretreatment) or cold Compound (1) (30-min pretreatment). The biodistribution of [11C]1 in a second species (Balb/c mice) showed a higher overall uptake of the radioligand with an average brain uptake of 8.9% dose/g. In C57BL/6-H3(−/−) knockout mice, a higher brain uptake was also observed. Analyses of metabolites and plasma protein binding were also undertaken. It appeared that [11C]1 could not specifically label H3 receptors in rodent brain in vivo. Possible causes are discussed.

Introduction

Histamine acts as a neurotransmitter in the central nervous system (CNS), regulating its own release and synthesis via a G-protein-coupled H3 autoreceptor [1], [2], [3], [4]. The H3 receptor also acts as a heteroreceptor, regulating the release of several other neurotransmitters, including dopamine [5], serotonin [6], noradrenaline [7], [8], glutamate [9], GABA and acetylcholine [10], [11]. In the mammalian brain, the highest H3 receptor densities have been found in the cerebral cortex, nucleus accumbens, striatum, olfactory bulb, substantia nigra and tuberomamillary nucleus [12], [13], [14]. Since the cloning of the H3 receptor [15], several H3 receptor isoforms have been discovered, displaying distinct regional CNS distribution and showing some differences in pharmacology and signaling [16], [17], [18], [19]. In addition, H3 receptors have shown significant differences in pharmacology between species [20], [21].

The abundance and localization of H3 receptors in the brain suggest that they may play a role in integrative nervous system functions and behaviors, such as in arousal mechanisms, regulation of food intake, and cognition and memory processes [22], [23], [24]. Thus, H3 receptor antagonists may have therapeutic potential in the treatment of sleep disorders and obesity [25], [26], and attention deficit hyperactivity disorder and Alzheimer's disease [27], [28].

Positron emission tomography (PET) is a noninvasive imaging method that can be used for studying CNS disorders [29], [30]. To further study the role of H3 receptors in the CNS and to evaluate the H3 receptor occupancy of potent H3-receptor-binding pharmaceuticals, a suitable H3 receptor PET radioligand would be highly beneficial [31], [32], [33]. Although a large number of selective H3 compounds have been discovered, no ligand suitable for PET imaging has been reported [34], [35], [36], [37], [38].

In selecting a proper H3 PET ligand, we considered the following requirements for the selection of our target compound: high selectivity and affinity for H3 receptor with fast on and off kinetics to the receptor and low nonspecific binding to tissues. In addition, the ligand should be sufficiently lipophilic to penetrate the blood–brain barrier, and it should be possible to rapidly synthesize it with high specific radioactivity [39]. 4-[3-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine [JNJ-10181457 (1)] is an H3 receptor antagonist, which has high selectivity and nanomolar affinity for its target, and has a benzylic carbon available for labeling (Fig. 1). In this paper, we report the radiosynthesis of [11C]1 and the evaluation of its potential as a radioligand for H3 receptor PET imaging.

Section snippets

Materials

1-[4-(3-Bromophenyl)-but-3-ynyl]-piperidine (3) was synthesized at Johnson & Johnson Pharmaceutical Research and Development LLC (San Diego, CA; unpublished data). All reagents used for radiosynthesis were purchased from Aldrich Chemicals. Anhydrous tetrahydrofuran was prepared by distillation over LiAlH4. High-performance liquid chromatography (HPLC) solvents were purchased from J.T. Baker (HPLC grade). 11CO2 was produced in an IBA 18/9 Cyclone cyclotron [40].

The semipreparative HPLC system

Radiosynthesis

Labeled compound [11C]1 was prepared from the starting material Compound (3) via a five-step synthesis route (Scheme 1), starting with Br–Li exchange reaction and carboxylation with 11CO2, followed by quenching with HCl [45]. Acid (3-(4-(piperidin-1-yl)but-1-ynyl)benzoic acid, 5) was treated with oxalyl chloride, after which the reaction mixture was evaporated to dryness. Amide (morpholine(3-(4-(piperidin-l-yl)but-l-ynyl)phenyl)methanone, 7) was prepared by adding morpholine to the residue,

Discussion

[11C]1 was prepared in a one-pot synthetic procedure in 28±8% (decay-corrected) yield, affording 1543±394 MBq of formulated product at the end of synthesis with high specific radioactivity (56±26 GBq/μmol), within a total synthesis time of 67±4 min.

The brain uptake of [11C]1 was evaluated in vitro, through autoradiography of coronal rat brain sections, and in vivo, through biodistribution studies in rats, Balb/c mice and C57BL/6-H3(−/−) mice. Two different H3 receptor antagonists, Compound (2)

Acknowledgments

We thank Dr. Eva de Rijke for mass spectral measurements, Dr. X. Langlois for preparing the brain sections for autoradiography and Ms. Marijke Stigter-van Walsum for her invaluable assistance in mice studies. We also thank Peter van Leuffen and the operators of BV Cyclotron VU for providing [11C]CO2.

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    Current address: Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska Hospital, S-17176 Stockholm, Sweden.

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