Background

[PubMed]

The neurotransmitter serotonin (5-HT) plays a major role in a variety of brain functions, such as appetite, sleep, and mood. Neuropsychiatric disorders, including major depression, schizophrenia, Alzheimer’s disease, and Parkinson’s disease (1-3), involve a dysfunction of the brain’s 5-HT system.

The serotonergic neurons – present in wide areas of the brain, including the raphe nuclei, hypothalamus, thalamus, and cerebral cortex – bear a protein called “serotonin transporter” (SERT) (4).The SERT, located on the cell bodies and terminals of 5-HT neurons, is a specific marker for the number and integrity of presynaptic terminals of serotonin-producing neurons. It regulates neurotransmission by removing released 5-HT from the extracellular space back into the presynaptic neuron. Commonly prescribed antidepressants are selective serotonin reuptake inhibitors, and their effects are obtained through interaction with (and inhibition of) the SERT (5). For that reason, in vivo imaging of the regional brain distribution of the SERT is an important tool for studying the 5-HT system and the treatment of neuropsychiatric disorders.

A variety of in vivo radioligands for positron emission tomography (PET) have been evaluated for imaging the SERT. trans-(+)1,2,3,5,6,10b-Hexahydro-6-(4-([¹¹C]methylthio)-phenyl)pyrrolo-(2,1-a)-isoquinoline ([¹¹C]McN5652) was the first successful and widely used agent (6, 7). However, it does have some limitations; for example, its kinetics in the brain are slow, and although adequate for regions with high SERT density, it often provides insufficient signal-to-noise differentials for imaging brain regions with intermediate to low SERT densities (e.g., limbic and neocortical regions) because of its high nonspecific binding. Over recent years, new PET radioligands have been synthesized and evaluated as SERT imaging agents and alternatives to [¹¹C]McN5652 (e.g [¹¹C]DASB, [¹¹C]AFM, [¹¹C]ADAM, [¹¹C]HOMADAM).

[¹¹C]McN5652 has two optical isomeric forms. The ability of its geometric trans (+), pharmacologically active enantiomer, [¹¹C](+)McN5652, to block the SERT is at least two orders of magnitude higher than that of its inactive enantiomer, [¹¹C](-)McN5652 (8, 9) (inhibition constant (K_i) ~ 0.7 nM for [¹¹C](+)McN5652 versus 0.4 nM for [¹¹C](-)McN5652) (also see the In Vitro Studies section). Brain uptake and retention of [¹¹C](+)McN5652 correspond to the distribution of the SERT in rodents, non-human primates, and humans. Several methods have been used to quantify [¹¹C](+)McN5652-specific binding, but the high level of nonspecific binding, the differences in nonspecific binding among regions, and the slow equilibration between specific and nonspecific binding populations have made such a quantification difficult (10).

Synthesis

[PubMed]

In 1995, Suehiro et al. (11) reported details of a synthetic procedure for [¹¹C]McN5652 that addressed the instability issue of the thiol precursor used in previous established synthesis methods (12). This improved procedure involved the saponification of thioesters and enabled reaction of the precursor of McN5652 in a one-pot radiolabeling sequence. After hydrolysis of the thioester functionality (via tetrabutylammonium hydroxide for 10 min), the free thiol was reacted in situ with [¹¹C]iodomethane (in dimethyl formamide, at 40-45 °C, for 1 min) without purification. The radiolabeled material produced was stable and could be used for multiple experiments.

The radiochemical yield obtained for [¹¹C]McN5652 was ~26% (decay-corrected at end of bombardment), and the total synthesis time, as reported by Suehiro et al. (11), was about 18 min. The specific activity of the radiotracer produced was ~84,730 MBq/μmol (2,290 mCi/μmol).

Details of a synthetic method for [¹¹C](+)McN5652 by S-methylation of the corresponding precursor with [¹¹C]iodomethane using an automated process were reported by Sasaki et al. (13). In this method, the desmethyl precursor was obtained by demethylation of nonradioactive McN5652 and successive stabilization by the addition of dithiothreitol (DTT), a protecting agent for the SH group, immediately after reaction. This method led to a radiochemical purity of 98.6 ± 0.4% and specific activity for [¹¹C](+)McN5652 of 181.3 ± 7.4 GBq/μmol (4,900 ± 200 mCi/μmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro inhibition studies performed by Shank et al. (9) showed that (+)McN5652 had the ability to prevent 5-HT uptake to a much greater extent than norepinephrine, whereas its potency for dopamine uptake was negligible. Its inhibition constant was found to be in the nanomolar range (K_i = 0.40 nM for synaptosomal 5-HT uptake), which was similar to those of paroxetine and sertraline, two high-affinity ligands for 5-HT. The optically inactive McN5652 enantiomer exhibited a much lower affinity (~150 lower) for the SERT than its optically active isomer (9).

Animal Studies

Human Studies

[PubMed]

The first studies of the serotonin 5-HT transporter in the living human brain, using PET with [¹¹C](+)McN5652, were reported by Szabo et al. (18). The experimental protocol involved injecting either [¹¹C](+)McN5652 or [¹¹C](-)McN5652 into three healthy subjects, and in two cases, injecting [¹¹C](+)McN5652 after pretreatment with the 5-HT uptake site blocker fluoxetine. Results showed highest accumulation of [¹¹C](+)McN5652, with a steady increase over 120 min after injection of the radiotracer. In contrast, with [¹¹C](-)McN5652 and when [¹¹C](+)McN5652 binding was inhibited with fluoxetine, radioactivity concentrations declined after reaching a peak at 20 to 30 min post injection.

Reivich et al. (19) used [¹¹C](+)McN5652 as a PET agent to study alterations in the brain 5-HT system in patients with depression. In their experiments, four drug-free depressed patients and four healthy control subjects received intravenous injections containing 7-10 mCi of [¹¹C](+)McN5652, and 18 sequential PET scans were obtained at increasing time periods. Distribution volume (DV) ratios of [¹¹C](+)McN5652 (compared with the cerebellum) were calculated in selected regions of interest, using a two-compartment model (20). Results showed significantly larger DV ratios in the left frontal cortex (P = 0.013) and the right cingulate cortex (P = 0.043), compared with healthy controls. Mean region/cerebellum DV ratios were as follows: 1.941 (in patients) and 1.667 (in control subjects) for the right thalamus, 1.678 (patients) and 1.153 (controls) for the right pons, and 1.359 (patients) and 1.092 (controls) for the right cingulate.

PET studies comparing [¹¹C](+)McN5652 and [¹¹C]cyanoimipramine were performed by Takano et al. (21) in 15 healthy volunteers. [¹¹C](+)McN5652 was injected at a dose of 179-759 MBq (4.8-20.5 mCi), and [¹¹C]cyanoimipramine was injected at a dose of 360-721 MBq before PET scans were performed. The following mean thalamus/cerebellum, striatum/cerebellum, and frontal cortex/cerebellum ratios were obtained at 90 min post injection: 1.61 ± 0.15, 1.59 ± 0.10, and 1.08 ± 0.07 for [¹¹C](+)McN5652 and 1.72 ± 0.18, 1.72 ± 0.18, and 1.17 ± 0.12 for [¹¹C]cyanoimipramine. After pretreatment with 50 mg of clomipramine (injected dose, 370 MBq (100 mCi)), the DVs of [¹¹C](+)McN5652 in the cerebellum and frontal cortex were not significantly changed, but those in the thalamus and striatum were dramatically decreased. The mean thalamus/cerebellum, striatum/cerebellum, and frontal cortex/cerebellum ratios for [¹¹C](+)McN5652 obtained at 90 min post injection were 1.20 ± 0.06, 1.31 ± 0.05, and 0.98 ± 0.03, respectively.

Over recent years, a number of human PET studies using [¹¹C](+)McN5652 have been performed to evaluate the neurotoxic effects of (±)3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”) on human brain 5-HT system (22-24). In a study by McCann et al (23). using [¹¹C](+)McN5652 and [¹¹C]DASB, global reductions in SERT concentrations were found in MDMA users with both PET ligands. [¹¹C](+)McN5652 has also been used in PET studies for quantifying the SERT in patients with bipolar disorder (21) and social phobia (25).

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