In vitro and in vivo characterisation of [11C]-DASB: a probe for in vivo measurements of the serotonin transporter by positron emission tomography

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Abstract

3-Amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, labeled with carbon-11 ([11C]-DASB), is a recently introduced radiotracer for imaging the serotonin transporter (SERT) by positron emission tomography (PET). A series of in vitro and in vivo experiments were performed to further characterise the properties of [11C]-DASB as an in vivo imaging agent for SERT. In vitro binding assays confirmed that DASB binds specifically to SERT with nanomolar affinity and high selectivity over a large number of other receptors, ion-channels and enzymes in the central nervous system. Ex vivo, [11C]-DASB binding in rat brain was shown to be saturable (ED50 of 56 nmoles/kg), and sensitive to both the number of available SERT binding sites and the number of viable serotonin neurons. Estimates of the radiation dose in man were extrapolated from rat biodistribution data (effective dose 5.5 E-03 mSv/MBq; critical organ -urinary bladder wall). Together with previous studies, the present findings indicate that [11C]-DASB is a very useful radiopharmaceutical for probing changes in SERT densities using PET imaging in the living human brain.

Introduction

The pursuit of a suitable radiotracer for in vivo imaging of the serotonin transporter (SERT) has occupied many groups over the past decade. Until recently, results have been indifferent at best. Most of the tested radiotracers suffered from low specific to non-specific binding ratios, lack of selectivity for SERT over other monoamine transporter proteins, inappropriate pharmacokinetics, or a combination thereof [2]. The most promising agent was (+)-McNeil 5652, labeled with carbon-11 [15] which has found use in positron emission tomography (PET) studies in both non-human primates [17] and humans [16]. Of late, a new class of compounds have been developed as putative radiotracers for in vivo imaging of SERT, namely the diarylthioethers [12], [21]. Iodinated congeners of the diarylthioethers, labeled with iodine-123, have found use in single photon emission computed tomography (SPECT) studies of SERT in non-human primates [1].

Recent studies have shown that the diarylthioether, [11C]-DASB ([11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, Figure 1) is a highly promising radiotracer for imaging the SERT by PET. [11C]-DASB and closely related analogues are efficiently and reliably synthesized from [11C]-iodomethane and the corresponding N-normethyl precursor [19], [20], [21]. In vitro binding experiments have demonstrated that DASB has high affinity for cloned human SERT and low affinity for the other monoamine transporters [20], while biodistribution studies in rats have shown that [11C]-DASB has a high brain uptake with a regional brain distribution consistent with the known distribution of SERT [3] and an in vivo brain pharmacokinetic profile befitting a SERT binding ligand. For example, at 60 min post injection hypothalamus (a SERT rich region) to cerebellum (a region essentially devoid of SERT) ratios of ca. 9:1 were obtained [20]. The promise of these results has been by realized by successful PET imaging of SERT with [11C]-DASB in cats [5], non-human primates [8] and humans [6], [7], [11]. PET imaging of SERT in the living human brain with [11C]-DASB has shown that SERT binding can be successfully modeled by a variety of non-invasive techniques, that an arterial input function is not required to derive an index of SERT densities, and that occupancy studies in patients and normal subjects treated with selective serotonin re-uptake inhibitors (SSRIs) is feasible [6], [11].

In the present work we sought to characterize further the properties of [11C]-DASB as an in vivo imaging agent for SERT. Our principal goal was to establish the usefulness of [11C]-DASB in a variety of paradigms applicable to imaging of SERT in the human brain. To this end, we completed four series of projects. The first established the selectivity of DASB using a broader array of in vitro binding assays, the second demonstrated the sensitivity of [11C]-DASB specific binding ex vivo in rat brain to SERT occupancy levels by SSRIs, and the third assessed the viability of [11C]-DASB as a neuronal marker of SERT in vivo. Fourthly, data obtained from the whole body distribution in rats is provided and extrapolated to estimate the human radiation dose upon [11C]-DASB administration.

Section snippets

Materials and methods

[11C]-DASB was synthesized as previously reported using the “Loop” method [19]. Purification was modified in that the HPLC eluent used was changed to 50/50 THF/H20 + 0.1N ammonium formate at 6 ml/min. Specific activities at end-of-synthesis were 35–60 GBq/μmole with radiochemical purities greater than 98%. (+)-McNeil 5652 was obtained from the National Institute of Mental Health’s Chemical Synthesis and Drug Supply Program. All other chemicals were obtained from commercial sources. All animal

In vitro selectivity

Previous in vitro and in vivo studies strongly indicated that regional brain uptake of [11C]-DASB in rat is mediated by binding to SERT [20]. In order to further establish this, more extensive in vitro binding assays of DASB were conducted. Two additional assays to measure the selectivity of DASB in the monoaminergic reuptake systems were carried out, namely competitive displacement studies of radiolabelled DAT, NET, and SERT selective ligands in rat brain tissue and competitive inhibition of

Conclusions

The results presented here and described previously indicate that [11C]-DASB is an excellent radiotracer for imaging SERT using PET. In vitro, DASB binds with high affinity and selectivity to only SERT out of a variety of other CNS binding sites including the other monoamine transporter proteins. Ex vivo in rat brain, [11C]-DASB specific binding displays appropriate pharmacology, saturability, and regional distribution with high signal to noise ratios. Significantly, levels of specific binding

Acknowledgements

We would like to thank Armando Garcia, Ruiping Guo, Lisa Richardson, Kevin Chueng, and Janice Shaw for their technical assistance. This work was financially supported by Eli Lilly (Canada).

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