High-resolution imaging of brain 5-HT1B receptors in the rhesus monkey using [11C]P943

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Abstract

The serotonin 5-HT1B receptors regulate the release of serotonin and are involved in various disease states, including depression and schizophrenia. The goal of the study was to evaluate a high affinity and high selectivity antagonist, [11C]P943, as a positron emission tomography (PET) tracer for imaging the 5-HT1B receptor. [11C]P943 was synthesized via N-methylation of the precursor with [11C]methyl iodide or [11C]methyl triflate using automated modules. The average radiochemical yield was approx. 10% with radiochemical purity of >99% and specific activity of 8.8±3.6 mCi/nmol at the end-of-synthesis (n=37). PET imaging was performed in non-human primates with a high-resolution research tomograph scanner with a bolus/infusion paradigm. Binding potential (BPND) was calculated using the equilibrium ratios of regions to cerebellum. The tracer uptake was highest in the globus pallidus and occipital cortex, moderate in basal ganglia and thalamus, and lowest in the cerebellum, which is consistent with the known brain distribution of 5-HT1B receptors. Infusion of tracer at different specific activities (by adding various amount of unlabeled P943) reduced BPND values in a dose-dependent manner, demonstrating the saturability of the tracer binding. Blocking studies with GR127935 (2 mg/kg iv), a selective 5-HT1B/5-HT1D antagonist, resulted in reduction of BPND values by 42-95% across regions; for an example, in occipital region from 0.71 to 0.03, indicating a complete blockade. These results demonstrate the saturability and specificity of [11C]P943 for 5-HT1B receptors, suggesting its suitability as a PET radiotracer for in vivo evaluations of the 5-HT1B receptor system in humans.

Introduction

Functional abnormalities of the 5-HT system have been linked to a variety of psychiatric disorders, including anxiety [1], [2], [3] depression [4], and schizophrenia [5], [6]. The serotonin 5-HT1B receptors, located on the 5-HT neurons, are autoreceptors involved in the regulation of synaptic 5-HT concentrations [7]. As a result, these receptors are a target for pharmaceutical development for their potential in the treatment of multiple psychiatric disorders, including anxiety, depression and substance abuse [8], [9], [10], [11]. It has been suggested that selective blocking of the 5-HT1B receptors in the brain could pave the way for a novel approach for managing psychiatric disorders [6], [12]. For example, in animal models, blockade of the terminal 5-HT1B receptors with an antagonist has been demonstrated to increase synaptic 5-HT concentration [13], [14]. In light of these observations, 5-HT1B antagonists, either alone or in combination with selective serotonin reuptake inhibitors (SSRIs), may hold potential as rapid- onset antidepressants. Thus, imaging the 5-HT1B receptors in vivo will aid in the elucidation of 5-HT1B receptor functions, and in the development of novel antidepressants.

P943, R-1-[4-(2-methoxy-isopropyl)-phenyl]-3-[2-(4-methyl-piperazin-1-yl)benzyl]-pyrrolidin-2-one, is a potent 5-HT1B antagonist in vitro, which binds with high affinity to the human 5-HT1B receptor expressed in HeLa cells (Ki=0.77 nM). The affinity of P943 for other 5-HT and non-5-HT receptors is at least 100-fold lower, making this a highly 5-HT1B-selective ligand (e.g. Ki=85 nM for 5-HT1A receptors) [15], [16].

Its specificity and selectivity was characterized by in vitro autoradiography studies, showing localization of radioactivity in regions known to contain high levels of the 5-HT1B receptor (e.g., globus pallidus and substantia nigra). Specific binding to these slices was demonstrated by co-incubation of the radiotracer with 10 μM serotonin. No specific accumulation was observed in the cerebellum, a region known to be devoid of 5-HT1B receptors. Specific displacement of the radiotracer was achieved using a structurally unrelated, but selective 5-HT1B inhibitor (CP-448,187). Its antagonist disposition was demonstrated in vitro in guinea pig substantia nigra, by blocking the reduction of 5-HT1B agonist-induced adenylate cyclase activities. The enantioselectivity of P943 has also been shown via ex vivo autoradiography studies in guinea pig [15], [16]. The details of these in vitro and in vivo screening and comparative studies will be published elsewhere. A summary of the in vitro pharmacology profile for unlabeled P943, which was performed by the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program, is shown in Table 1, demonstrating its potency and selectivity towards 5-HT1B receptors.

The aim of the present study was to carry out pre-clinical evaluation of [11C]P943 in non- human primates with the high resolution research tomography (HRRT) positron emission tomography (PET) scanner. For initial quantitative evaluation, the binding potential (BPND) was calculated using the equilibrium ratios of regions to cerebellum, assuming this region is devoid of 5-HT1B receptors. The saturability and specificity of the tracer binding was assessed by blocking studies with unlabeled parent compound and GR127935 (a selective 5-HT1B/5-HT1D antagonist). Here, we report the preparation of [11C]P943 and its evaluation in non-human primates to assess its in vivo characteristics.

Section snippets

General

Reagents and solvents were purchased from the standard commercial sources, i.e., Sigma-Aldrich, Fisher Scientific, Merck or J. T. Baker, and were used without purification. Both the P943 standard and the N-desmethyl-precursor were provided by Pfizer.

Synthesis of [11C]P943 was carried out either with a TRACERLab FxC automated synthesis module (GE Medical Systems), or with a AutoLoop (Bioscan, Washington, DC, USA). For the FxC module, the radioligand was purified by a preparative high-performance

Chemistry

[11C]P943 contains a piperazine side chain that is common in 5-HT1B antagonists [11]. N-Alkylation of the normethylpiperazine moeity of the pyrrolidinone precursor 1 (1-(4-(2- methoxypropan-2-yl)phenyl)-3-(2-(piperazin-1-yl)benzyl)pyrrolidin-2-one) was carried out with [11C]methyl triflate in DMF at 100°C for 4 min (Fig. 1, Method A) in the FxC module. Theaverage radiochemical yield was aprox.10±2% at the end of synthesis (based on trapped [11C]methyl triflate) with a specific activity of

Discussion

P943 is a 5-HT1B receptor antagonist with physiochemical properties suitable for use as a PET radiotracer. A series of preclinical and clinical assessments performed at Uppsala Imanet, Sweden confirmed that [11C]P943 is a suitable tracer for in vivo evaluations of the 5-HT1B receptor system [15], [16]. Here we report the radiolabeling of [11C]P943 that was carried out at the Yale PET Center using automated synthesis modules. Both the GE TRACERLab FxC automated synthesizer and Bioscan AutoLoop

Conclusion

[11C]P943 is a suitable ligand for PET imaging studies of 5-HT1B receptors in vivo. This ligand has been advanced to imaging applications in humans and it will be a useful new tracer for studying the functional significance of 5-HT1B receptors in humans and its role in various disease states.

Acknowledgments

The authors thank the staff of the Yale University PET Center for their technical expertise and support and Jean-Dominique Gallezot, Ph.D., for helpful discussions. The authors also thank the NIMH Psychoactive Drug Screening Program for the in vitro assays of P943. Funding for this study was provided by the Yale Pfizer Bioimaging Alliance. This publication was also made possible by CTSA Grant Number UL1 RR024139 from the National Center for Research Resources (NCRR), a component of the National

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    Current address: Kreitchman PET Center, Columbia University Medical Center, New York, NY.

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