Original Articles
Radiosynthesis and biodistribution of 123I-labeled antagonists of the histamine H3 receptor as potential SPECT ligands

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Abstract

We have synthesized three 123I-labeled histamine H3 receptor ligands, i.e., [123I]GR 190028, [123I]FUB 271, and [123I]iodoproxyfan, in moderate to good radiochemical yields via a Cu+-assisted I-for-123I exchange method. Biodistribution in the rat of these compounds revealed high hepatic and pulmonary uptake. Brain uptake was moderate, but for [123I]iodoproxyfan, brain uptake was high enough for a pilot single photon emission computed tomography (SPECT) study in the rabbit. However, for this compound, the cerebral uptake could not be blocked by a pretreatment with [R]-α-methylhistamine, a selective, high-affinity histamine H3 receptor agonist, both in the SPECT study in the rabbit and in the biodistribution study in the rat. Apparently, [123I]iodoproxyfan is binding to a non-H3 receptor binding site. None of the three investigated compounds is suitable for use as a SPECT ligand for the H3 receptor in the brain.

Introduction

Histamine acts as a neurotransmitter in the central nervous system (CNS) via three receptor subtypes, the H1, H2, and H3 receptor (22). The H3 receptor, the most recently discovered subtype, acts as an autoreceptor regulating release and synthesis of histamine in histaminergic neurons (1). Moreover, the H3 receptor also acts as a heteroreceptor and influences the release of other neurotransmitters such as noradrenaline (19), serotonin (4), acetylcholine (5), and dopamine (18). Because of these characteristics, expectations are high regarding the H3 receptor as a target for the development of drugs for the therapy of several CNS disorders 10, 27.

To further elucidate the relation between the H3 receptor and neuronal deficiencies in terms of localization and density of the H3 receptor, a noninvasive in vivo method to investigate these parameters is required. Single photon emission computed tomography (SPECT) is such a noninvasive method that can provide the information needed if a suitable radioligand is available. A potential SPECT radioligand for the H3 receptor can be developed if the following requirements are fullfilled. Besides having a selective, reversible, and high-affinity binding, a selected compound also should preferably be an antagonist, because the H3 receptor is most likely to be a G-protein-coupled receptor (3) and therefore the receptor will have two affinity states for an agonist, and this will influence the binding characteristics of a potential SPECT ligand. Furthermore, a potential SPECT ligand for the H3 receptor must penetrate into the brain after systemic administration and therefore must have a better in vivo pharmacological profile than thioperamide.

The first published radioiodinated H3 antagonist, [125I]-iodophenpropit 1 8, 12, was also labeled with 131I for biodistribution studies in rats. However, this compound showed poor brain uptake, probably because the isothiourea moiety is positively charged at physiological pH. The development of iodophenpropit as a potential SPECT ligand for brain imaging after labeling with 123I was therefore terminated (7). The lack of brain penetration also ended the development of three potential positron emission tomography (PET) ligands for the H3 receptor, [18F]VUF 5000 (30), [18F]FUB 272, and [11C]UCL 1829 (17).

As has been mentioned, for a potential SPECT ligand for the imaging of the H3 receptor in the CNS, it is preferable to know in advance whether this compound is taken up by the brain or not. Ex vivo binding studies can provide such information. However, it has been shown that thioperamide (Fig. 1) crosses the blood–brain barrier (BBB) 15, 25, but the closely related [18F]VUF 5000 does not (30). These results suggest that care must be taken with information from ex vivo binding experiments, because this information can be misleading, probably due to altered pharmacokinetic properties of compounds when administered in tracer amounts, as is done in SPECT studies.

It has been shown by Clitherow et al. (2) that GR 175737, a 1,2,4-oxadiazole histamine H3 receptor antagonist (Fig. 1), is more potent in an ex vivo H3 receptor binding assay as compared with thioperamide, while being less potent in an in vitro H3 receptor assay (Table 1). An explanation for this finding could be that GR 175737 crosses the BBB more readily than thioperamide. This difference is an indication that the iodinated analogue, GR 190028, will also cross the BBB more readily than thioperamide, bearing in mind that the substitution of chlorine by iodine will hardly influence this characteristic. Although GR 190028 does not have a high affinity for the H3 receptor such as iodoproxyfan ( Ki=6 nM [2], Bmax=40–150 fmol/mg [7], theoretical B/F=0.03), the compound was still selected as a potential SPECT ligand because it was anticipated that GR 190028 had a relatively good brain penetration.

Another iodinated H3 receptor ligand known in literature is iodoproxyfan (24). Iodoproxyfan was claimed to be an antagonist of the H3 receptor, but recent studies have suggested that iodoproxyfan is a partial agonist of the H3 receptor 20, 29. Altough a potential SPECT ligand for the H3 receptor should preferably be an antagonist, as has been stated above, iodoproxyfan was still selected because the in vitro pharmacology has been studied extensively. Iodoproxyfan was radiolabeled with 125I 9, 24 and in vitro autoradiography in the rat brain using [125I]iodoproxyfan showed the known distribution of the H3 receptor. This binding could be blocked almost completely in the presence of 1 μM of [R]-α-methylhistamine (11). The authors also studied the binding of [125I]iodoproxyfan in rat striatum membranes. They found a Bmax of 78 fmol/mg and a Kd of 65 pM, which results in a B/F ratio of 1.2.

FUB 271 is an analogue of iodoproxyfan that is closely related chemically (Fig. 1), and was presented recently as an antagonist of the H3 receptor (23, 24. Although FUB 271 is less potent in vitro than iodoproxyfan ( Ki=7 nM [24], Bmax=30–190 fmol/mg [7], theoretical B/F=0.03), a structurally related chloro derivative (FUB 181) showed high in vivo CNS activity (23). Because of these results with FUB181, it was anticipated that FUB 271 would penetrate much better into the brain than thioperamide; therefore, we selected FUB 271 as a potential SPECT ligand.

The aim of this study was to radiolabel GR 190028, iodoproxyfan, and FUB 271 with 123I and to evaluate these radiolabeled compounds for their use as SPECT ligands.

Section snippets

General procedure for radiolabeling with 123I (13, 14)

To a solution of 5.0 mg (22 μmol) of 2,5-dihydroxybenzoic acid, 10 mg (52 μmol) citric acid, 20 μL of a 13-mM CuSO4 solution in water, and 0.20 mg (10 μmol) SnSO4 in 450 μL of 10% (v/v) ethanol/water, and ca. 250 μg of substrate were added. This mixture was shaken until all compounds were dissolved.

123I was produced in-house according to the 124Xe (p,2n)123Cs → 123Xe → 123I nuclear reaction. 124Xe gas (99.9% enriched) was bombarded with 24 MeV protons, generated by a Philips AVF30 cyclotron and

Results and discussion

The histamine H3 receptor antagonistic activity of GR 190028 and FUB 271 has been determined at the guinea pig jejunum and is given in Table 1. GR 190028 and FUB 271 are both potent H3 receptor antagonists, whereas iodoproxyfan behaves as a partial H3 receptor agonist (see above) 20, 29 and not an antagonist as reported earlier for other bioassays 11, 24.

Besides its biological activity, another important characteristic of a potential brain imaging PET ligand is its LogDoct,7.2 value. This

Conclusion

We have radiolabeled three histamine H3 ligands with 123I and evaluated these compounds for their potential use as SPECT ligands for brain imaging of the H3 receptor. Biodistribution revealed that only [123I]iodoproxyfan penetrated the brain sufficiently well to allow a pilot SPECT study in the rabbit. The subsequent study showed a high uptake of [123I]iodoproxyfan in the basal ganglia, which, however, could not be blocked by the selective H3 receptor agonist [R]-α-methylhistamine. The uptake

Acknowledgements

P.J. van Leuffen and the operators of the Philips cyclotron are greatly acknowledged for the production of 123I. Dr. P.J. Beswick of Glaxo Wellcome Medicines Research Centre is acknowledged for the generous gift of GR 190028. Ms. M.-T. Pakbiers is acknowledged for operating the Siemens MultiSPECT 3. The research of Dr. R. Leurs was made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.

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