Background

[PubMed]

Serotonin (5-HT) is now known to serve as the gastrointestinal signaling molecule and participate in the development of alcohol dependence (1, 2). It is also involved in a variety of physiological functions such as the post-meal satiety process, as well as the pathogenesis of Alzheimer’s and Parkinson’s diseases, anxiety, and depression (1-3). Imaging and data obtained by the analysis of resected tissue of epileptic patients and studies in animal models have provided much evidence that endogenous 5-HT, the activity of its receptors, and pharmaceuticals with 5-HT agonist and/or antagonist activities are believed to be involved in the pathogenesis of epilepsy (4). Thus, imaging of the 5-HT system (SERT) is considered important to understand a variety of neurological and psychiatric functions and disorders.

A variety of drugs have been labeled with radioactive iodine (¹²³I), carbon (¹¹C), or fluorine (¹⁸F) for imaging of the 5-HT receptor or transporter using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) (5). In general, these compounds are either nonselective or have a low specificity for SERT (6). Subsequently, a new drug, 2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine (ADAM), radioiodinated with ¹²³I or ¹²⁵I, has been evaluated by several investigators for imaging of SERT.

Synthesis

[PubMed]

The synthesis of ADAM has been detailed by Oya et al. (7). A mixture of 2,5-dibromonitrobenzene, 2-thio-N,N,-dimethylbenzamide, and potassium carbonate in dimethylformamide was heated at 100°C for 24 h. The mixture was added to cold water, and the precipitates were collected and recrystalized from ethanol. This reaction produced a light yellow solid, 2-(4-bromo-2-nitrophenylthio)-N,N-dimethylbenzamide, with a yield of 79%. The light yellow solid was dissolved in tetrahydrofuran (THF), and a mixture of borane (BH₃) in THF was added to it. The mixture was refluxed for 2 h and stirred overnight at room temperature. The solvent was removed under reduced pressure to obtain a residue. Water was added to the residue, and the mixture was refluxed for 30 min with vigorous stirring. The solution was neutralized (pH 7–8) with sodium bicarbonate and extracted three times with a mixture of dichloromethane and methanol. The extractions were combined and dried over sodium sulphate, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, and a colorless oil, 2-(4-bromo-2-nitrophenylthio)-benzyl)dimethylbenzamide, with a yield of 83%, was obtained.

The nitro group was reduced in a mixture of HCl, methanol, and stannous chloride at 10°C. With this reaction a residue of 5-bromo-2-((2-((diethylamino)methyl)phenyl)thio)phenylamine was obtained with a yield of 78%. This residue was dissolved in triethylamine, and bis(tributyltin) and tetrakis(triphenylphosphine)palladium were added to it. The mixture was heated to 100°C in a sealed bottle for 48 h, and the solvent was subsequently removed under reduced pressure. A residue of 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(tri-n-butyltin)-phenylamine was obtained as a colorless oil and purified on preparative silica gel plates. The yield of this reaction was 59%.

The colorless oil was dissolved in chloroform, and a solution of iodine in chloroform was added drop-wise until the iodine color persisted. The solution was stirred overnight at room temperature, and a solution of potassium fluoride in methanol was added. A solution of sodium bisulphate was added to the mixture and stirred for 5 min. The organic layer was separated and dried over sodium sulphate, and the solvent was removed under reduced pressure. The final product, ADAM, which is a colorless oil, was purified on preparative silica gel plates. This reaction had a yield of 99%.

To prepare radioiodinated ADAM, ¹²³I/¹²⁵I-labeled sodium iodide in 1 N HCl was mixed with 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(tri-n-butyltin)-phenylamine in a sealed vial, and hydrogen peroxide was added through a syringe at room temperature. The iodination reaction was terminated after 10 min by the addition of saturated sodium persulphate, and the resulting solution was neutralized with sodium bicarbonate solution. The mixture was extracted with ethyl acetate, and the organic layer (purity, 90%; yield, 94%) was dried over sodium sulphate. The solvent was then removed under a stream of dry nitrogen, and the residue was purified by high-performance liquid chromatography. The radiochemical purity of the product was 99%. The specific activities of the compounds were not reported by the investigators, although the stability of [¹²⁵I]ADAM was reported to be 3 months under refrigerated conditions. The corresponding [¹²³I]ADAM was reported to be stable for 24 h after labeling. Another group of investigators who prepared [¹²³I/¹²⁵I]ADAM by a similar method reported the specific activity to be 8.88 × 10⁶ GBq/mmol (240,000 Ci/mmol) for [¹²³I]ADAM and 8.14 × 10⁴ GBq/mmol (2,200 Ci/mmol) for [¹²⁵I]ADAM (8).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Using rat frontal cortical membrane homogenates for saturation binding studies with [¹²⁵I]ADAM, the radiochemical had a dissociation constant (K_d) of 0.15 ± 0.03 nM and a B_max of 194 ± 65 fmol/mg protein (8). In another study, the binding affinity (K_i) of [¹²⁵I]ADAM toward SERT on cloned LLC-PK1 cell membranes (of pig kidney origin) was 0.013 nM with a >1,000 fold selectivity over the respective norepinephrine (K_i = 699 nM) and dopamine (K_i = 840 nM) transporters.

Animal Studies

Human Studies

[PubMed]

SPECT was used in two laboratories to investigate the biodistribution of [¹²³I]ADAM in humans (6, 16). An analysis of the combined data from the two studies revealed that the radiolabel accumulated in the MB with a distribution that was consistent with selective binding to SERT (16). The region/CB ratios for the SPECT scans were 1.95 ± 0.13 for the MB, 1.27 ± 0.10 for the medial temporal regions, and 1.11 ± 0.07 for the ST. The investigators concluded that [¹²³I]ADAM was a safe and suitable radiochemical for SERT imaging in the human brain. In a preliminary study, the binding of [¹²³I]ADAM was investigated in human patients with major depressive disorder and normal healthy controls (17). Results from this study suggested that, compared to the control individuals, there was a decreased binding of the label in the MB region of the major depressive disorder patients that correlated with the severity of the depressive symptoms. The investigators also observed an age-related decrease in binding of the label in the MB SERT of the control subjects.

Using healthy volunteers, Booij and de Win as well as other investigators reported that the optimal imaging time for [¹²³I]ADAM SPECT was ~5 h after a single injection of the radiochemical (18-20).

To determine the possible involvement of a SERT-related endophenotype for purging or non-purging bulimia nervosa (BN), the binding of [¹²³I]ADAM was investigated in female twins (21). The purging BN women were observed to have a higher binding of the label in the MB compared to the non-purging BN women and the normal controls. The investigators concluded that, because this study was performed in a small group (13 BN patients), the results should be interpreted with caution and the observations should be verified with a study involving a larger population. In another study, it was shown there was a significant (P < 0.001) increase in brainstem SERT availability in individuals suffering from migraines (22). However, the investigators suggested that whether SERT played a direct or an indirect role in the development of this condition remains to be determined.

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