Background

[PubMed]

The serotonin neurotransmitter is considered to play an important role in a variety of brain functions such as appetite, sleep, and mood. Neuropsychiatric disorders, including major depression, schizophrenia, and Alzheimer’s and Parkinson’s diseases (1-3), involve a dysfunction of the brain’s serotonin system. The serotonergic neurons – present in wide areas of the brain, including the hypothalamus, thalamus, and cerebral cortex – bear a protein called ”serotonin transporter” (SERT) (4).

The SERT, located on the cell bodies and terminals of 5-hydroxytryptamine (5-HT) neurons, is a specific marker for the number and integrity of presynaptic terminals of serotonin-producing neurons. The SERT regulates neurotransmission by removing released serotonin from the extracellular space back into the presynaptic neuron. Commonly prescribed antidepressants are selective serotonin reuptake inhibitors (SSRIs), and their effect is 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 in the treatment of neuropsychiatric disorders.

A variety of in vivo radioligands for positron emission tomography (PET) have been evaluated for imaging the SERT. [¹¹C]McN5652 was the first successful and widely used agent (6, 7), but it does have some limitations because of its slow brain kinetics and low to nonspecific binding ratios in humans. It is adequate for regions with high SERT density but often provides insufficient signal-to-noise differentials for imaging brain regions with intermediate to low SERT densities (e.g., limbic and neocortical regions).

Over recent years, new PET radioligands have been synthesized and evaluated as SERT imaging agents. Among them are ¹¹C-labeled N,N-dimethyl-2-(2-amino-4-cyanophenylthio)benzylamine ([¹¹C]DASB), 2-(2-(dimethylaminomethyl)phenylthio)-5-fluoromethylphenylamine ([¹¹C]AFM), 5-bromo-2-[2-(dimethylaminomethylphenylthio)]phenylamine ([¹¹C]DAPA), 2-[2-(dimethylaminomethylphenylthio)]-5-iodophenylamine ([¹¹C]ADAM), and N,N-dimethyl-2-(2´-amino-4´-hydroxymethylphenylthio)benzylamine ([¹¹C]HOMADAM), which are based on a diaryl sulfide motif (4). [¹¹C]2-(2-((Dimethylamino)methyl)phenylthio)-5-(2-fluoroethyl)phenylamine ([¹¹C]AFE) has shown a high binding affinity and good selectivity for the SERT and displays signal-to-noise ratios that may enable more reliable mapping of brain regions with low SERT density.

Synthesis

[PubMed]

[¹¹C]AFE can be prepared from its monomethylamino precursor 5-(2-fluoroethyl)-2-(2-((methylamino)methyl)-phenylthio)phenylamine by reaction with either [¹¹C]methyl triflate ([¹¹C]MeOTf) (8) or [¹¹C]MeI, as described by Zhu et al. (9).

In the first method (with [¹¹C]MeOTf), the precursor is dissolved in acetone and [¹¹C]MeOTf is bubbled through the solution at room temperature. Once maximum activity is reached, the mixture is heated (70 °C for 5 min in a water bath) and purified by high-performance liquid chromatography (HPLC; 40% acetonitrile–60% 0.1 ammonium acetate, pH 6.8). The product is then eluted (~12 min), diluted in 100 ml of H₂O, loaded on a SepPak, and then eluted with ethanol. The ethanol solution is then mixed with 0.9% sterile saline and filtered through a 0.22 μm membrane to obtain the final product, [¹¹C]AFE.

In the second method, [¹¹C]AFE is prepared via reaction of the precursor with [¹¹C]MeI (trapped at –10 °C). The reaction is performed at 80 °C for 5 min.

The reported radiochemical purity of [¹¹C]AFE produced by the first method is >95% (determined by HPLC). The reported radiochemical yields at the end of the synthesis are 32 ± 17% (decay-corrected, based on [¹¹C]MeOTf; n = 6) in the first method (8) and 26 ± 14% (decay-corrected, based on [¹¹C]MeI; n = 4) in the second method (9). The specific activities of the radiotracer produced are 61,790 ± 32,190 GBq/mmol (1,670 ± 864 Ci/mmol) and 32,079 ± 16,502 GBq/mmol (867 ± 446 Ci/mmol) for the first (8) and second methods (9), respectively.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The affinities of [¹¹C]AFE for the monoamine transporters SERT, dopamine transporter (DAT), and norepinephrine transporter (NET) were determined in vitro by Zhu et al. (9). Experiments were performed with cloned human receptors transfected on HEK-293 cells and the radioligands [³H]paroxetine (for SERT), [³H]nisoxetine (for NET), and [³H]GBR2935 (for DAT). Experiments were performed according to a previously published procedure (10).

[¹¹C]AFE displayed a high affinity for SERT (K_i = 1.80 ± 0.07 nM), but much lower affinities for NET (K_i = 946.2 ± 222.5 nM) and DAT (K_i > 10⁴ nM). Additional assays showed that [¹¹C]AFE displayed virtually no affinity for benzodiazepine, opioid (μ, σ, and κ) receptors, or for serotonin and dopamine receptors such as 5-HT_1A, 5-HT_2a, 5-HT_2c, 5-HT₃, 5-HT₆, 5-HT₇, and D₁-D₅.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

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Zhu Z , Guo N , Narendran R , Erritzoe D , Ekelund J , Hwang DR , Bae SA , Laruelle M , Huang Y . The new PET imaging agent [11C]AFE is a selective serotonin transporter ligand with fast brain uptake kinetics. Nucl Med Biol. 2004;31(8):983–994. [PubMed: 15607480]

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